IMPROVING ADENOVIRUS-BASED VACCINE EFFICACY BY THE INNATE
IMMUNE MODULATORS EAT-2 AND REA
By
Yasser Ali Aldhamen
A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Microbiology and Molecular Genetics
2012
ABSTRACT
IMPROVING ADENOVIRUS-BASED VACCINE EFFICACY BY THE INNATE
IMMUNE MODULATORS EAT-2 AND REA
By
Yasser Ali Aldhamen
Despite recent advances, there is a great need for more potent formulations to enhance
immunogenicity of vaccines. Pro-active induction and/ or harnessing of beneficial innate
immune responses may be the mechanism underlying the effectiveness of certain adjuvants to
significantly contribute to the ability of vaccines to generate adaptive immune responses. Several
classes of immune receptors have been targeted for novel adjuvant development. An important
emerging class of immune receptors is the SLAM family of receptors. We endeavored to develop
a strategy to improve the efficacy of vaccines by incorporation of proteins known to be important
in SLAM mediated signaling. In this dissertation, we described that coexpression of the SLAMꞌs
adaptor, EAT-2, along with pathogen derived antigens facilitates induction of beneficial innate
immune responses, resulting in improved induction of antigen-specific adaptive immune
responses. We utilized recombinant Adenovirus-based vaccines expressing murine EAT-2 along
with HIV-1/Gag or Plasmodium falciparum derived circumsporozoite (CS) protein. As
compared to appropriate controls, rAd5 vectors expressing EAT-2 facilitated bystander
activation of NK-, NKT-, B-, and T-cells early after their administration into animals. EAT-2
expression also augments the expression of surface maturation markers of antigen presenting
cells (CD40, CD80, CD86, MHC-II, and CCR7). Indeed, this multi-tiered activation of the innate
immune system by vaccine mediated EAT-2 expression enhanced the induction of antigen
specific cellular immune responses. We also compared the utility of vaccine mediated
ii
expression of EAT-2 relative to another innate immune stimulator, a recombinant TLR agonist
derived from Eimeria tenella, referred to as rEA. We confirmed that rEA activates multiple
immune cell types and elicits the induction of pleiotropic pro-inflammatory cytokines in a
MyD88-dependent manner. Surprisingly, we also found that the TRIF adaptor protein acts as a
potent negative regulator of TLR agonist-triggered immune responses. However, we uncovered a
potent immunosuppressive activity inherent to the combined expression of the CS protein and
rEA, in contrast to continued enhancement of CS specific adaptive immune responses by use of
the EAT-2 adjuvant.
We subsequently went on to partially unveil the mechanism underlying the ability of the
EAT-2 adaptor protein to regulate the induction of adaptive immune responses. We demonstrate
that EAT-2 expression specifically prevents vaccine induced CRACC upregulation on APCs in a
MAPK-dependent mechanism. Confirming these results, utilization of a mutated SH-2 domain
form of EAT-2 adaptor (EAT-2(R31Q) failed to prevent CRACC upregulation. We have also
demonstrated that EAT-2 expression triggers the production of several cytokines and
chemokines from macrophages in a MAPK-dependent and independent mechanisms.
Future studies will further expand upon these findings. We leave our readers with the
view that, understanding the molecular mechanism and the role of SLAM/ EAT-2 and rEA
adjuvant activity, will allow for the development of next generation vaccine platforms for use in
a number of immunotherapy approaches targeting HIV-1 and Malaria derived antigens, as well
as numerous other vaccine targets in general.
iii
DEDICATION
I dedicate this dissertation to my wonderful family. Particularly to my understanding and
patient wife, Suha, who has put up with these many years of research and to our precious
daughter Seba and son Ahmed, who are the joy of our lives. Also, I would like to dedicate this
doctoral dissertation to my parents. There is no doubt in my mind that without their continued
support and counsel I could not have completed this process.
iv
ACKNOWLEDGEMENTS
I would like to gratefully and sincerely thank my mentor Dr. Andrea Amalfitano for his
guidance, understanding, patience, and most importantly, his friendship during my graduate
studies at Michigan State University. I have been amazingly fortunate to have an advisor who
gave me the freedom to explore on my own, Dr. Amalfitano constantly supported and
encouraged me to develop scientific thinking, writing skills, critical data analysis and planning of
experiments. I am not sure many graduate students are given the opportunity to develop their
own individuality and self-sufficiency by being allowed to work with such independence. I
specifically thank Dr. Amalfitano for supporting and supervising the rAd5-EAT2 project, a
project that completely transformed my research here at Michigan State University. For
everything you’ve done for me, Dr. Amalfitano, I thank you. I hope that one day I would become
as good an advisor to my students as Dr. Amalfitano has been to me.
I greatly appreciate the help provided by my guidance committee: Dr. Norbert Kaminski,
Dr. Sungjin Kim, Dr. Kefei Yu, and Dr. Ian York. Their insightful comments and constructive
discussions have significantly improved my research progress as well as development of critical
scientific thinking.
I also thank my friend and co-worker, Dr. Sergey Seregin, who has been always there to
listen and give advice. Sergey and I performed tremendous amount of experiments working with
our novel Ad vectors, Ad5-DAF and Ad5-EAT2. I am deeply grateful to him for the long
discussions that helped me sort out the technical details of my work. I am also thankful to him
for encouraging and helping me to build Ad5-EAT2 vaccine vector.
I also thank Dr. Daniel Appledorn. Dr Appledorn's insightful comments and constructive
criticisms at different stages of my research were thought-provoking and they helped me focus
v
my ideas. I am grateful to him for helping and supporting my research as well enforcing strict
validations for each research result, and thus teaching me how to do research.
My sincere thanks to all current and former members of Dr. Amalfitano lab: Dr. Charles
Aylsworth, Sarah Godbehere Roosa, Nathaniel Schuldt, Aaron McBride, Tyler Voss, Joyce Liu,
Youssef Kousa, Dionisia Quiroga, David Rastall, William Nance, William De Pas, Megan
Hoban, Jenny Zehnder, Brandy Burke. All of them are genuinely nice and eager to help each
other, and I’m glad, I have worked and interacted with them.
My special thanks to Sarah Godbehere Roosa, who is a wonderful technician and
exceptionally pleasant person to work with.
Special thanks to all MSU core facilities, including: ULAR and, in particular, all
employees of BPS/biochemistry animal housing facility; MSU Histopathology labs; MSU
Genomics Technology Support Facility: Flow Cytometry (Dr. Louis King), quantitative RT-PCR
(Jeff Landgraf). Without their great performance and skilled assistance our research would not
have been possible.
I’m thankful to all faculty members and employees of the Department of Microbiology
and Molecular Genetics, especially the Chair of the department (Dr. Walter J. Esselman) and the
Director of MMG Graduate program (Dr. Robert Hausinger) for their kindness and full support
provided during these years.
Most importantly, none of this would have been possible without the love and patience of
my family. My family, to whom this dissertation is dedicated to, has been a constant source of
love, concern, support and strength all these years. I would like to express my heart-felt gratitude
to my family. My family has aided and encouraged me throughout this endeavor. I warmly
vi
appreciate the generosity and understanding of my family. Finally, I appreciate the financial
support from The Ministry of Higher Education of Saudi Arabia, King Abdullah Bin Abdulaziz
Scholarship Program.
vii
TABLE OF CONTENTS
List of Tables……………………………………………………………….…....xvi
List of figures……………………………………………………………………xvii
List of Abbreviations………………………………………………………….....xxi
Chapter 1
1.1
1.2
1.2.1
1.2.2
1.3
1.3.1
1.3.2
1.4
1.5
1.6
1.6.1
1.6.2
1.7
1.7.1
1.7.2
Chapter 2
2.1
2.1.1
2.1.2
2.1.3
2.1.3.1
2.1.3.2
Introduction…………………………………………...…………1
Innate immune system…….…………………….…………...…..2
Adaptive immunity and immunological memory …………….....4
T cells activation and cellular immunity...…………................…6
B cell activation and humoral immunity……………….………..12
Regulation of adaptive immunity by the innate immune
system……………………………………………………………15
Innate immune cell-dependent regulation of the adaptive
immune system…………………………………………………..16
The impact of innate immune recognition on adaptive immune
cell responses………………………………………………….....21
Vaccine Adjuvants………………………………………………23
The immuno-modulatory molecule: recombinant Eimeria
tenella derived antigen (rEA……………………………………..24
Harnessing innate immunity by the SLAM family of receptors
adaptor EAT-2…………………………………………………...26
The signaling lymphocytic activation molecules (SLAM)
family ofreceptors………………………………………………..26
Signal transduction of SLAM family of receptors by SAP
family of adaptors………………………………………………..28
Adenoviruses as vaccine vectors………………………………....31
Molecular basis for cellular recognition of Adenovirus
Vectors……………………………………………………………33
Adaptive immune responses to Ad vector expressed
transgenes………………………………………………………..35
Expression of the SLAM family of receptors adaptor EAT-2
as a novel strategy for enhancing beneficial immune
responses to vaccine antigen………………………………..........37
Introduction……………………………………………………...38
Human immunodeficiency virus (HIV) and AIDS
global epidemic…………………………………………………..39
HIV genetic diversity and its impact for HIV control…………...40
Immune response to HIV-1 and immune evasion mechanisms….41
Innate immune responses to HIV-1……………………………...41
Adaptive immune responses to HIV-1…………………………..45
viii
2.1.4
2.2
2.3
HIV-1 vaccine Studies………………………………………..…48
Results…………………………………………….……..……...55
Discussion…………………………………………….……..…..89
Chapter 3
Vaccine platforms combining Circumsporozoite protein and
potent immune modulators, rEA or EAT-2, paradoxically
result in opposing immune responses …………………..…….…93
Introduction……………………………………………………...94
Immune responses to Plasmodium parasites…………………….95
Malaria vaccine development……………………………………97
Results …………………………………………………………..103
Discussion…………………..…………………………………...134
3.1
3.1.1
3.1.2
3.2
3.3
Chapter 4
4.1
4.2
4.3
Chapter 5
5.1
5.2
5.3
Chapter 6
6.1
6.1.1
6.1.2
6.1.3
6.1.4
6.2
6.2.1
6.2.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
6.10
6.11
TRIF is a critical negative regulator of TLR agonist mediated
activation of dendritic cells in vivo …….………………..…..…..140
Introduction…………………….………………………….…….141
Results…………………………………………………………...144
Discussion……………………………………………………….165
Preventing CRACC receptor upregulation in antigen presenting
cells improves induction of antigen specific adaptive immune
responses by vaccines.……….……… ………………………….173
Introduction…………...…………………………………………174
Results………………....…………………………………………177
Discussion………………….…………………………………….200
Material and Methods…………………………..………………....207
Adenovirus vector construction…………………… ……….........208
EAT-2 expressing Ads construction……………..….……..……..208
Ad-HIV/Gag construction…………………………..……………209
Ad-CSP construction……………………………….…….............209
Ad-GFP and Ad-GFP/rEA construction……………..….……….209
Validation of viral particles (VP) titers of Ads………..………....210
Silver Staining…………………………………………..………..210
Western Blotting………………….…………………..…….........210
Animal procedures……………………….………….…….……..211
Cytokine and chemokine analysis……………………..…………212
Quantitative RT-PCR Analysis………………………..…………212
Isolation of Splenocytes…………………..……………..……….214
Cell staining and flow cytometry………………………..……….215
In vitro cell culture………………………………………..……....216
Murine CD11c+ DCs isolation……………………………..……..217
Murine IL12p70 measurement by ELISA from isolated
CD11c+ DCs……………………………………………......…….217
CD8+ T cells depletion Analysis………………….……………....218
ix
6.12
6.13
6.14
6.15
6.16
ELISPOT Analysis………………………………………………..218
In vivo CTL Assay………………………………………………...219
Detection of CSP antibody in murine serum by ELISA…….....….220
Western blotting…………………………………………………...220
Statistical analysis………………………………………………....221
Chapter 7
Overall summary and significance ………………………………..223
Bibliography………………………………………………………………..……..230
x
LIST OF TABLES
Table 1
Table 1: Expression pattern of SLAM family of receptors
in hemopoietic cells………...……………………………………..27
Table 2
The SAP family of SH-2 domain containing adaptors…………….29
Table 3
List of primers, utilized in qRT-PCR experiments………………..214
xi
LIST OF FIGURES
Figure 1
Systemic administration of EAT-2 expressing adenovirus vector
induces cytokine and chemokines responses………….…..............57
Figure 2
Transduction efficiency of innate immune cells by Adenovirus
vectors expressing transgenes………….………………………….59
Figure 3
Ad-EAT2-mediated activation of innate and adaptive immune
cells in vivo………….………………………...…………………..61
Figure 4
Ad-EAT2-mediated activation of innate and adaptive immune
cells in vivo………………………….…………………………….63
Figure 5
IFNγ production from NK cells 6 and 48 hours after Ads
injection…………………………………………………………...65
Figure 6
Ad-EAT2-mediated activation of innate and adaptive
immune cells in vivo………………………………………………67
Figure 7
HIV-Gag specific cellular immune responses elicited by
Ad-HIV/Gag and Ad-EAT2 co-immunization……………………70
Figure 8
HIV-Gag specific cellular immune responses elicited by
Ad-HIV/Gag and Ad-EAT2 co-immunization……………………72
Figure 9
Analysis of the breadth of Gag-responses…………........................74
Figure 10
Analysis of T cell epitope responses of Balb/c and C57Bl/6
mice to HIV-Gag in Ad-HIV/Gag and Ad-EAT2
co-injected mice…………………………………………………...76
Figure 11
Cellular immune responses after CD8+ T cells depletion in
Ad-HIV/Gag and Ad-EAT2 co-immunized mice……………..…..78
Figure 12
Ad-HIV/Gag and Ad-EAT2 co-immunization increases
the frequency of HIV-Gag specific CD8+ T cells………................81
Figure 13
Increased cytolytic activity of the Gag-specific T-cell
in vivo in Ad-HIV/Gag and Ad-EAT2 co-immunized mice……....84
Figure 14
EAT2 overexpression augments CD80 and CD86
expression by bone marrow derived macrophages…………..........86
xii
Figure 15
Increased expressions of CD40, CD80, CD86, and MHC-II in
Ad-EAT2 infected RAW264.7 cells………….................................88
Figure 16
CS protein sequence……………………….....................................99
Figure 17
Ad-CSP construction………….......................................................104
Figure 18
Ad-CSP Stimulates CS protein specific T and B cell responses….107
Figure 19
TLR agonist rEA induced innate cytokines 6 hours
post injection………………………………………………………109
Figure 20
Immuno-modulating proteins conversely affect IFNγ secreting
splenocytes………….......................................................................111
Figure 21
CS protein expression does not interfere with antigen specific
immune responses against other transgenes at low doses………....113
Figure 22
Ad-GFP/rEA combined with 5x107 vp/mouse of Ad-CSP
begins to display a diminished CS protein specific CMI
response after a dose of 5×106 vp/mouse…………………............115
Figure 23
Co-expression of CS protein and EAT-2 stimulates more
potent CS protein specific CMI responses…………………...........118
Figure 24
Expression of GFP does not interfere with CS protein specific
CMI responses……………………………......................................120
Figure 25
Co-expression of CS protein and EAT-2 increases the
breadth of response against CS protein………………...................123
Figure 26
Improved degranulation of CD8+ T cells in mice
co-vaccinated with Ad-CSP and Ad-EAT2………………………125
Figure 27
Co-expression of CS protein and EAT-2 increases
cytolytic activity of CS protein specific T cells……......................127
Figure 28
Induction of CS protein specific antibody responses
by Ad-CSP vaccines augmented by rEA or EAT-2………………129
Figure 29
Sub-isotype analysis of IgG antibody from plasma of mice
co-vaccinated with Ad-CSP and Ad-EAT2…………....................131
Figure 30
CD3+ CD8- IFNγ+ cells respond similarly to both
vaccine regimens………………………………………………....133
xiii
Figure 31
TRIF acts as a negative regulator of rEA-induced
MyD88-dependent activation of dendritic cells in vivo…………..146
Figure 32
TRIF acts as a negative regulator of rEA-induced
MyD88-dependent activation of macrophages in vivo……………148
Figure 33
TRIF acts as a negative regulator of rEA-induced
MyD88-dependent activation of dendritic cells
in vivo (MFI) ……………………………………………………...150
Figure 34
TRIF acts as a negative regulator of rEA-induced MyD88dependent activation of NK, NKT, T, and B cells in vivo………...153
Figure 35
TRIF negatively regulates rEA-mediated MyD88 dependent
activation of pro-inflammatory cytokines and chemokines
in vivo………..................................................................................156
Figure 36
TRIF negatively regulates rEA-mediated MyD88
dependent activation of pro-inflammatory cytokines and
chemokines in dendritic cells……………………………………...159
Figure 37
TRIF negatively regulates cytokine production by DCs,
triggered by several common TLR agonists………….....................162
Figure 38
rEA-triggered Erk1/2 phosphorylation is MyD88
dependent. C57BL/6 WT or MyD88-KO mice were
injected with 100 ng of rEA……………………………………….164
Figure 39
TRIF acts as a negative regulator of rEA-induced signaling
and downstream responses in DCs: model of action……………..170
Figure 40
EAT-2 functions as a negative regulator of Adenovirus
mediated induction of CRACC receptor expression on
macrophages in vitro……………………………………………………179
Figure 41
EAT-2 over-expression induces similar transcript
levels of innate immune responses genes as compared
to adenovirus control………….......................................................181
Figure 42
EAT-2 transcript levels and virus protein quantification
by BCA……………………………………………………………183
Figure 43
EAT-2 over-expression reduces protein level of CRACC
receptor on DCs and macrophages in vitro………….....................186
Figure 44
EAT-2 over-expression negatively regulates CRACC
xiv
expression in dendritic cells in vivo……………...……….............188
Figure 45
Mutant form of EAT-2 adaptor does not prevent
CRACC upregulation by Adenoviruses…………………………..191
Figure 46
EAT-2 requires functional ERK and PLCγ pathways
to downregulate CRACC receptor on APCs…………...................194
Figure 47
EAT-2 over-expression induces ERK phosphorylation………….196
Figure 48
EAT-2 is a critical cytokine and chemokines regulator
in macrophages……………………………………………...........199
Figure 49
Model of EAT-2 molecular mechanism in APCs………………...205
xv
LIST OF ABBREVATIONS
Ad
Adenovirus
ADAR
Adenosine deaminase-RNA-specific (IFN-inducible)
AIDS
Acquired immunodeficiency syndrome
ALL
Acute lymphocytic leukemia
ALT
Alanine aminotransferase
ANOVA
Analysis of variance
AP-1
Activator protein-1
APC
Antigen presenting cell
ASC
Apoptosis- associated Speck-like Protein Containing
a Caspase Recruitment Domain
AsGM1
Anti-asialo GM1
ATCC
American type culture collection
BCR
B cell receptor
BMDMs
Bone marrow derived macrophages
Bcl-2
B-cell lymphoma 2
CAR
Coxsackie and adenovirus receptor
C3
Complement component 3
CEA
Carcinoembryonic antigen
CFSE
Carboxyfluorescein succinimidyl ester
CMI
Cell mediated immunity
CMV
Cytomegalovirus
xvi
CR
Complement receptor
CRACC
CD2-like receptor activating cytotoxic cells
CRAD
Conditionally replicative adenoviruses
CSP
Circumsporozoite protein
CTL
Cytotoxic T lymphocyte
CTLA-4
Cytotoxic T-Lymphocyte Antigen 4
CXCL-9
Chemokine, induced by IFN
DAF
Decay accelerating factor
DAI
DNA-dependent activator of interferon regulatory factors
DAMP
Danger associated molecular pattern
DCs
Dendritic cells
DNA
Deoxyribonucleic acid
dpi
Days post injection
DR5
Death receptor
EAT-2
Ewing's sarcoma-associated transcript-2
EC
Endothelial cells
Env gp120
Envelope glycoprotein 120
EDTA
Ethylene-diamine-tetra-acetic acid
EGTA
Ethylene-glycol-tetra-acetic acid
ELISA
Enzyme-linked immunosorbent assay
ELISPOT
Enzyme-linked immunosorbent spot assay
EM
Electron microscopy
Erk1/2
Extracellular Signal-Regulated Kinases 1 and 2
xvii
ERT
EAT-2-related transducer
FACS
Fluorescence-activated cell sorting
FBS
Fetal bovine serum
Foxp3
forkhead box P3
GAPDH
Glyceraldehyde 3-phosphate dehydrogenase
GFP
Green fluorescent protein
G-CSF
Granulocyte colony-stimulating factor
GM-CSF
Granulocyte-macrophage colony-stimulating factor
GMP
Good manufacturing practice
FcγRIIB
Fc gamma receptor IIb
FDA
Food and drug administration
HA
Hemagglutinin
HBV
Hepatitis B virus
HBsAg
Hepatitis B viral surface protein
HDAd
Helper-dependent adenovirus
HEK293
Human embryonic kidney 293
HIV
Human immunodeficiency virus
HLA
Human leukocyte antigen
HMGB-1
High-mobility group box 1 protein
hpi
Hours post injection
HSV-1
Herpes simplex virus-1
HVR
Hypervariable region of Ad hexon protein
ICAM-1
Inter-Cellular adhesion molecule 1
xviii
ICS
Intracellular staining
IFN, IFN
Interferons and (type I IFNs)
Ig
Immunoglobulin
IL-
Interleukins (pro-inflammatory cytokines)
IM
Intramuscular
IP
Intraperitoneal
IP-10
IFN-γ inducible protein 10
IPS1
IFN-b promoter stimulator-1
IRF3, IRF7,
Interferon Regulatory Factors 3 and 7
ISG
Interferon stimulatory gene
ITAMs
Immunoreceptor tyrosine-based activation motifs
ITSMs
Immunoreceptor tyrosine based switch motifs
IV
Intravenous
JAK-1, JAK-3
Janus kinases 1 and 3
KC (CXCL-1)
Keratinocyte derived chemokine, murine analog of human IL-8
KO
Knockout
LPS
Lipopolysaccharides
Ly-9, Ly-108
Lymphocyte antigen 9, 108
MAL
MyD88-adaptor-like
MAPK
Mitogen-activated protein kinases
MadCAM-1
Mucosal addressin cellular adhesion molecule-1
MHC
Major histocompatibility complex
ml
Milliliter
xix
MPEC
Memory precursor effector cells
MPER
Membrane proximal external region
mM
Milli-molar
mg
Milligram
l
Micro-liter
M
Micro-molar
g
Microgram
MCP-1 (CCL-2)
Monocyte chemotactic protein 1
MCMV
Murine cytomegalovirus
MIP-1 (CCL-4)
Macrophage inflammatory protein 1 beta
MyD88
Myeloid differentiation factor 88 (TLR adaptor)
mTOR
Mammalian target of rapamycin
MFI
Mean fluorescent intensity
MOI
Multiplicity of infection
MPL
Monophosphoryl lipid A
MZ
Marginal zone
Nab
Neutralizing antibody
NALP3
NACHT, LRR and PYD domains-containing protein 3
Nef
Nuclear factor
NFκB
Nuclear factor kappa B
NHP
Non-human primates
NK
Natural killer
NKT
Natural killer T cell
xx
NLRs
Nucleotide-binding oligomerization domain/leucine-rich repeat
receptors
NOD-1, NOD-2
Nucleotide-binding oligomerization domains 1 and 2
NTB-A
Natural killer, T and B cell antigen
OD
Optical density
OTC
Ornithine transcarbamylase
PAMP
Pathogen-associated molecular pattern
PBMC
Peripheral blood mononuclear cells
PBS
Phosphate buffer saline
PCR
Polymerase chain reaction
PEG
Polyethylene glycol
PI3K
Phosphoinositide 3-kinase
PLCγ
Phospholipase C gamma
PLGA
Polyactic glycolic acid
PRR
Pattern recognition receptor
PSF
Penicillin, streptomycin, fungizone
qRT-PCR
Quantitative reverse transcriptase Polymerase chain reaction
RANTES
Normal T-cell expressed, and secreted
RCA
Replication competent adenovirus
rEA
Recombinant Eimeria tenella derived antigen
RES
Reticulo-endothelial system
RIG-1
Retinoid-inducible gene 1
RLR
RIG-1-like receptors
xxi
RNA
Ribonucleic acid
RORδt
Retinoic acid receptor related orphan receptor gamma t
SAP
SLAM-associated adaptors
SARM
Sterile α-and armadillo-motif containing protein
SD
Standard deviation
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel
Electrophoresis
SEM
Standard error of the mean
SFCs
Spot forming cells
SH-2
Src-homology 2
SIV
Simian immunodeficiency virus
SLAM
Signaling Lymphocytic Activation Molecules
SLEC
Short-lived effector cells
SP-A
Surfactant protein-A
TBK-1
TANK-binding kinase 1
TCID
Tissue culture infectious dose
TCR
T cell receptor
TEC
Thymic epithelial cells
TFH
Follicular T helper
TGF-β
Transforming growth factor beta
Th
T helper
TIR
Toll/Interleukin-1 receptor
TLR
Toll-Like Receptor
xxii
TNF
Tumor necrosis factor alpha
TRAIL
TNF-related apoptosis-inducing ligand
TRAM
TRIF-related adaptor molecule
TRIM
Tripartite motif
TRIF
Toll/Interleukin-1 receptor (TIR)-domain-containing adaptorinducing interferon-β
UV
Ultraviolet
VCAM-1
Vascular cell adhesion molecule 1
ViViD
Violet viability dye
VP
Viral particle
WT
Wild-type
xxiii
Chapter I
Introduction
1
1.1
The innate immune system:
The innate immune system is conserved across species and represents the first line of
general defense against pathogenic infections (1). Engagement and activation of specific
components of the innate immune system plays a critical role in the enhancement of T and B cell
responses of the adaptive immune system (2). Microbial detection by the innate immune system
relies primarily on receptors that recognize a wide range of specific molecular structures present
in many microbes known as “pathogen associated molecular patterns” (PAMPs). These PAMPs
are detected by the host’s deployment of a wide array of extracellular (secreted), cell surface, or
cytosolic molecules, proteins, and receptors generally known as “pattern recognition receptors”
(PRRs) (3-5). Unlike the antigen receptors of B and T cells that are somatically generated and
clonally distributed, the PRRs are encoded in the germline and not subject to somatic variations.
PRRs lack the specificity of the T and B cell antigen receptors. PRRs can also recognize
endogenous signals released during host cell stress or death, commonly known as “damageassociated molecular patterns” (DAMPs) (6). Secreted PRRs bind to microbial cell surfaces,
activate the complement system, and facilitate opsonization of pathogens for phagocytosis by
macrophages and neutrophils (7). The transmembrane PRRs include the toll-like receptors
(TLRs) family and the C-type lectins (8). The cytosolic PRRs include the nucleotide-binding
oligomerization domain/leucine-rich repeat receptors (NLRs) and the retinoic acid-inducible
gene (RIG)-1-like receptors (RLRs) (9).
The innate immune system is composed of a network of different cell types expressing or
reacting to PRR activation, including: monocytes/macrophages, dendritic cells (DCs), natural
killer (NK) cells, NKT cells, neutrophils, gamma delta (γδ) T cells, and mast cells. Each cell of
the innate immune system expresses various types of PRRs (9). In addition, the cells of the
2
adaptive immune system, both T and B cells, express multiple innate recognition receptors,
implying that pathogen recognition is sophisticated and involves orchestration between innate
and adaptive immune cells (10). Innate immune responses are non-specific and more rapid; they
occur within minutes to hours following infection, whereas the adaptive immune responses
usually take several days to weeks. However, compared to adaptive immune responses that last
for a long period of time, the innate immune responses rapidly wane as a result of multiple
negative feed-back mechanisms in order to limit the tissue damage that can result from these
potent responses (8).
The most widely studied and the best characterized family of PRRs are TLRs (4). The
transmembrane-located Toll receptor was first identified in the early 1980s in Drosophila. The
Drosophila Toll receptor was found to be required for responses to fungal and Gram-positive
bacterial infections (11). TLRs were discovered in mammals in mid-1990s. Their discovery gave
rise to a tremendous amount of studies, which shed light on TLRs signaling mechanisms
associated with the innate immune response. Thirteen mammalian TLRs (10 of which are found
in humans) have been identified to date.
TLRs are type I transmembrane proteins that variously contain: 1) an extracellular
domain containing leucine-rich repeats that mediate the recognition of PAMPs; 2) a
transmembrane domain; and 3) an intracellular Toll–interleukin 1 (IL-1) receptor (TIR) domain
required for downstream signal transduction (12). The expression of TLRs is cell-type specific,
and present in many cell types inclusive of non-hematopoietic epithelial and endothelial cells
(EC), macrophages, dendritic cells (DCs), natural killer (NK) cells, and neutrophils (2).
TLRs are either expressed on the plasma membrane or in the endosomal/ lysosomal
compartments (13). Plasma membrane TLRs recognize conserved cell surface PAMPs, such as
3
Lipopolysaccharide (LPS) of Gram-negative bacteria (TLR4), lipoteichoic acid of Gram-positive
bacteria and bacterial lipoproteins (TLR1/ TLR2 and TLR2/ TLR6), and flagelline (TLR5) (13).
Endosomal TLRs mainly detect microbial nucleic acids, such as double stranded RNA (dsRNA)
(TLR3), single stranded RNA (ssRNA) (TLR7), and dsDNA (TLR9) (13). Five TLRs signaling
adaptors have been identified including the Myeloid differentiation primary response gene (88)
MyD88, MyD88-adaptor-like (MAL/TIRAP), TIR-domain-containing adaptor protein inducing
interferon (IFN)-β (TRIF) (also known as TICAM1), TRIF-related adaptor molecule (TRAM)
(also known as TICAM2), and sterile α-and armadillo-motif containing protein (SARM) (14).
MyD88 is used by all TLRs except TLR3, MAL is used by TLR2 and TLR4, TRIF is used by
TLR3 and TLR4, and TRAM is used only by TLR4 (4). Once activated by a specific ligand, the
various TLR interactions with the TLR adaptor proteins trigger a series of intracellular signaling
cascades that result in down-stream activation of transcription factors such as nuclear factor
(NF)-κB and activated protein-1 (AP-1), leading to transcription of immune response genes and
production of pro-inflammatory cytokines and chemokines. In addition, activation of TLRs 3,
4,7,8, and 9 can also result in activation of the interferon regulatory factors (IRFs) 3 and/or 7
signaling pathways, leading to production of type I interferon (IFNs) responses that limit the
replication of invading pathogens, as well as the promotion and shaping of pathogen-specific Band T-cell adaptive immune responses (7, 15).
1.2
Adaptive immunity and immunological memory:
Unlike the innate immune system that provides critical mechanisms for the rapid sensing
and elimination of a wide range of invading pathogens, the adaptive immune system is a system
capable of specific recognition, of both self and nonself-antigens, and generation of
4
immunological memory (16). The typical functions of the adaptive immune system are primarily
carried out by two cell type: the effector cells, the T lymphocytes, and antibody-producing cells,
the B lymphocytes. T lymphocytes mature in the thymus from common lymphoid progenitors
derived from the bone marrow or fetal liver (17). In contrast, B lymphocytes mature primarily in
the bone marrow (18). Immature T and B lymphocytes go through a positive and a negative
selection process. The positive selection process evaluates the ability of antigen receptors of
immature lymphocytes to bind to peptide sequences in general, while negative selection
processes acts to identify and eliminate cells that are reactive to self-peptides/antigens.
Negative selection for immature B lymphocytes occurs in the bone marrow and spleen.
Binding to self-antigen in bone marrow leads to deletion, anergy, or receptor editing (18). B cells
that survive negative selection in the bone marrow migrate to the spleen, a place where any selfantigen reactive B cells become anergic and destroyed (19). Progenitor T lymphocyte selection
on the other hand, occurs in the thymus where thymic epithelial cells (TECs) play a critical role
in this process. Positive selection for T cell receptors (TCRs) occurs in the thymus cortex.
Negative selection to remove self-reactive immune responsive T cells occurs in the medulla (20).
These two selection processes leads to the formation of naïve lymphocytes, with the capability to
recognize peptide sequences from foreign, but not self, antigens. In this context, it is important
to note here that, negative selection is an incomplete process as it does not remove all selfreactive lymphocytes (21). Thus, a mechanism called peripheral tolerance exists to prevent
inappropriate responses by self-reactive lymphocytes that escaped negative selection. One
mechanism of peripheral tolerance is induction of anergy in T cells that recognize self-antigens
in the absence of co-stimulatory signals (22). Another mechanism of peripheral tolerance is
+
regulated by a subset of CD4 T-helper cells, called regulatory T cells (Treg), which suppress
5
inappropriate adaptive immune responses to self-antigens via production of immune suppressive
cytokines, such as IL-10 and TGF-β (23).
Naïve T and B lymphocytes are highly mobile. Following their development in the
primary lymphoid organs, they migrate to secondary lymphoid organs, including lymph nodes
and the spleen, and then to several sites in the body during infections. Lymphocyte trafficking is
facilitated by an array of several adhesion molecules. For example, α4β7 integrins (which binds
to mucosal addressin cellular adhesion molecule-1 (MadCAM-1) on gut epithelial cells)expressing lymphocytes, preferentially traffic to the gastrointestinal tract, whereas CLA-1CCR4-bearing lymphocytes home to the skin (24).
1.2.1 T cell activation and cellular immunity:
Mature T cells are activated via interaction of their TCR complex with antigenic peptides
+
complexed with MHC molecules on the surface of APCs. CD8 T cells can interact with
peptides (9-11 amino acids in length) presented on MHC class I molecules. These MHC class Irestricted peptides are generally derived from cytosolic proteins (self-proteins or proteins from
pathogens that replicate within the cells, such as viruses and intracellular bacteria). In contrast,
+
CD4 T cells interact with peptides derived from extracellular antigens (approximately 18-20
amino acids in length) presented on the MHC class II molecules. Unlike MHC class I molecules
that are constitutively expressed in all nucleated cells, MHC class II molecules are expressed
only on APCs in response to inflammatory stimuli, such as the ligands of TLRs (8). The TCR is
composed of an α and β protein heterodimer which is non-covalently linked to the signaltransducing CD3 complex. Signaling through the TCR is initiated by the activation of Src protein
tyrosine kinases leading to the phosphorylation of CD3 immunoreceptor tyrosine-based
6
activation motifs (ITAMs) followed by recruitment of ZAP-70 and activation of associated
+
adapter proteins (25). During infections, naïve CD8 T cells are primed by mature APCs in
secondary lymphoid organs, leading to their activation and clonal expansion. T cell activation
involves dramatic changes in T cell metabolism, such as enhanced uptake of glucose, amino
acids, and iron (26). As a result, activated pathogen-specific T cells go through multiple rounds
of replication to generate enormous numbers of effector memory T cell population, a population
that represent the first line of defense of the adaptive immune response (27).
Several receptors and inflammatory mediators have been implicated as necessary to
prime naïve T cells for effector functions. For example, signals produced from the TCR-MHC
interaction, costimulatory molecules (derived from interactions between other cell surface
molecules (such as CD28, SLAM, OX-40, and CD27), and inflammatory cytokines receptors
(such as IL-12 and type I IFN), activate several signaling pathways (such as, the PI3K-PDK1Akt-mTOR signaling pathway) and result in T cell proliferation and expansion (28). This process
is sometimes as referred to as the three signal hypothesis. Type I IFNs (mainly IFNα) has been
+
shown to potently enhance CD8 T cell expansion and antigen specific cytotoxicity (29).
Further, IL-12, through induction of T-bet via an mTOR-dependent mechanism, has also been
+
shown to play a critical role in terminal differentiation of CD8 effector memory T cells (30). In
addition, besides its role as a T cell growth factor, IL-2 has been shown to also function as a
memory T cell differentiation factor by promoting the differentiation of effector memory cells
+
(31). For example, culturing naïve CD8 T cells in a high concentration of IL-2 facilitate
induction of superior effector functions, as compared to cells cultured in low concentrations of
7
IL-2 (32). In addition, the TLRs, IL-1, the IL-18 signaling adaptor protein, and MyD88 have also
been recently shown to play a role in effector T cell expansion and survival (33).
+
Once primed, effector memory CD8 T cells are capable of migrating to inflamed tissue
via expression of inflammatory cytokine receptors, such as CXCR3, that allow them to enter and
+
target to peripheral tissues (34). At the peripheral site of infection, effector memory CD8 T
cells kill the antigen expressing infected cells (via Granzyme B and Fas-mediated apoptotic
+
pathways) and release several cytokines, such as IFNγ, TNFα, and IL-2. Effector memory CD8
T cells have also been shown to have immunoregulatory function. For example, it has been
+
shown recently that a subset of effector memory CD8 T cells provide immunoregulatory signals
via the immunosuppressive cytokine, IL-10, to prevent excessive tissue injury at the site of
infection (35).
+
During the expansion phase, antigen-specific CD8 memory T cells go through many
phenotypic and functional changes, including, the re-expression of the IL-7 receptor α, CD127.
+
Effector memory CD8 T cells are characterized by increased expression of the CD127 (36).
Pathogen-specific memory T cells are induced during initial engagement with foreign antigens
and are divided into two populations: the short-lived effector cells (SLEC) (characterized by low
low
expression of ,CD127
) that mostly die off (90-95%) when the infection is cleared, and the
memory precursor effector cells (MPEC) that survive the contraction phase and contribute to
long term immunological memory populations (characterized by increased expression of
high
CD127
) (37). These CD127 expressing effector memory T cells (TEM) further differentiate
8
into self-renewing central memory T cells (TCM), called “memory stem-cells”, that can persist
for extended period of time in the absence of antigenic stimulation (38).
Several signaling pathways and cellular mediators have been described to play important
roles during the TEM to TCM transition. For example, the Wnt-β-catenin signaling pathway has
+
+
been shown to arrest effector CD8 T-cell differentiation and to promote development of CD8
memory stem cells (39). More recently, the specific inhibitor of mTOR, rapamycin, which has
been used extensively as an immunosuppressive drug during tissue transplantations, has been
+
shown to enhance memory CD8 T cell differentiation during vaccination, confirming a major
+
regulatory role for mTOR in driving central memory CD8 T cell differentiation (40).
Central memory T cells are characterized by increased expression of the anti-apoptotic
marker Bcl-2, re-expression of the lymph node homing receptors CD62L and CCR7, and higher
expression levels of CD127 (38). In addition, central memory T cells are also characterized by
enhanced recall responses to previously encountered antigens and higher protection capacity
compared to early-stage memory T cells (38). The survival of memory T cells is dependent on
the survival cytokines, IL-7 and IL-15, which maintain the memory cells in a state of slow but
continuous proliferation (41).
+
CD4 T cells are the other group of T lymphocytes. They constitute the largest portion of
the T cell population in the body. Most of these cells serve a helper function and are thus
designated as T helper (Th) cells. T-helper cells differentiate from naive to effector T cells, a
process regulated by interactions with antigen presenting DCs. Following their activation, CD4
+
T cells produce a wide range of cytokines that play critical roles in mediating adaptive immunity
9
+
to a variety of pathogens. Once activated, CD4 T cells facilitate antibody production by B
+
lymphocytes (18), enhance and maintain responses by CD8 T cells, regulate macrophage
function, enhance the potency of DCs, and regulate immune responses to control autoimmunity
and to adjust the magnitude and persistence of immune responses (42).
+
CD4 T cells are also classified into several effector cell subsets. Based on their
+
cytokine expression patterns or profile, CD4 T cells (both in mice and human) were initially
divided into two major subsets, designated Th1 and Th2 (43). The Th1 cell subset was
characterized by their ability to produce their signature cytokine, IFNγ. In contrast, Th2 cells
+
variously produce IL-4, IL-5, and IL-13. Differentiation of naïve CD4 T cells into effector Th1
type cells is influenced by IL-12 (produced by activated DCs) and IFNγ (produced by activated
NK and NKT cells) and is regulated by the T-box expressed in T-cells (T-bet) transcription
factor (42). In contrast, Th2 differentiation of these T cells is driven by IL-4 (which is produced
by activated T cells and other cells) and the transcription factor GATA-3 (42). Both Th1 and Th2
cells function to eliminate different pathogens in the body. Cytokines produced from Th1 cells
enhance cell-mediated immunity of NK cells, macrophages, and cytotoxic T cell to kill and
eliminate intracellular pathogens and virally stressed cells (44). Th2 cytokine profile has been
shown to enhance humoral immunity, thus helping in eliminating extracellular pathogens and
parasites (44). In addition, Th1 and Th2 cells have also been shown to differ in their homing
capabilities as a result of differences in the expression pattern of several chemokine receptors.
For example, Th1 cells express CCR5 and CXCR3, while Th2 cells express CCR4 and CCR3
receptors (44).
10
In the past few years, several additional subsets of T-helper cells have been described. A
subset designated as Th17 has been identified (45). Th17 cells produce IL-17 as their signature
cytokine as well as IL-22 and GM-CSF (44). Th17 cells are induced by IL-6 and TGF-β, express
the transcription factor RORγt (retinoic acid receptor related orphan receptor γt), have distinct
homing capabilities (based on CCR6 expression), and are required for the elimination of fungi
and extracellular bacteria (46). The existence of Th2 cells subset that produce high amounts of
the cytokine IL-9 in response to IL-4 and TGF- β, are designated as Th9 cells (47). In addition, a
subset of T-helper cells, designated Th22 cells, produce IL-22 and express the transcription
factor aryl hydrocarbon receptor (48). Th22 cells have been shown to play an important role in
the skin due to the expression of skin homing receptors, CCR10 and CLA, and the production of
IL-22, a stimulator of antimicrobial peptides by keratinocytes (48). Furthermore, a subset of Th
cells called follicular T helper (TFH) cells, that resides in the lymph node and spleen has also
+
been described. TFH cells are central memory CD4 T cells that express the chemokine receptor
CXCR5, which mediates their recruitment to follicles. TFH cells can produce both IL-4 and
IFNγ. TFH cells also enhance B cell activation (via T-cell-dependent antigen), which leads to
germinal center formation. This facilitates B cell proliferation, immunoglobulin isotype class
switching, and affinity maturation of antigen-specific B cells. These processes eventuate in the
generation of memory B cells and long-lived plasma cells that produce high affinity somatically
mutated antibodies of switched isotypes (49).
Another subset of CD4 T-helper cells, designated as Treg cells, selectively produce IL10,as well express the IL-2 receptor α chain (CD25) and the transcription factor forkhead box
protein 3 (foxp3) has also been described. Treg cells are divided into two main subsets: naturally
11
+
occurring Foxp3 regulatory T cells, which develop in the thymus, and inducible regulatory T
+
cells, which develop in the periphery from conventional CD4 T cells as a result of specific
stimuli, such as regulatory cytokines or immunosuppressive drugs (50). Inducible Treg cells are
further divided into three types including: T regulatory 1 (Tr1) cells, which secrete IL-10, TGF-β
+
producing Treg cells, and inducible Foxp3 regulatory T cells (51).
Treg cells suppress T cells responses by either direct or indirect mechanisms. Cell-cell
contact and/ or production of the immunosuppressive cytokines, IL-10 and TGF-β, can directly
inhibit effector T cells responses, whereas Treg cells can modulate the function of DCs and
thereby indirectly inhibit effector T cells responses (52). Several studies have indicated that
production of IL-10 and TGF-β by Treg cells suppresses host immune responses and induces
self-tolerance, thus limiting the magnitude of effector T cell responses and helping to minimize
collateral tissue damage caused by pathogen instigated immune responses (51). For example,
deletion or blockade of IL-10 leads to enhanced clearance of Leishmania major parasites in mice
(53). Also, reduced pulmonary inflammation and lung injury in a mouse model of Pneumocystis
pneumonia infection has been directly linked to the availability of Treg cells (54). Furthermore, it
has been shown that depletion of Treg cells prior to vaccination, enhanced anti-tumor immunity
and increased the antigen-specific T cell responses to vaccine antigens (55), implicating a critical
role for Treg cells in vaccination regimens.
12
1.2.2 B cell activation and humoral immunity:
B cell development is regulated by diverse signaling pathways and transcription factors
(18). B lymphocyte development and function are regulated by signals transduced through the Bcell antigen receptor (BCR). Engagement of the BCR results in activation of several protein
tyrosine kinase (PTK) signaling pathways including the Src-family PTKs (Lyn, Fyn, and Blk),
Syk, and the Tec-family member Btk (56). B cell development and function are also regulated by
signals transduced by other B-cell-specific cell surface molecules such as CD19, CD20, CD21,
CD22, CD23, CD24, CD40, Igα (CD79a), and Igβ (CD79b) (57). Each of these B cell-specific
cell surface molecules has distinct functions during B cell development and activation. For
example, CD19, which is expressed by all B cell lineages, functions to regulate intracellular
signal transduction by amplifying the Src-family kinase activity (57). CD19 functions as
membrane adaptor protein, which recruits the signaling molecules Vav, PI3K, and Lyn that
activate the PLCγ and MAPK signaling pathways (58). Several reports have shown that CD19CD21 complex is crucial for B cell function. For example, mice lacking either CD19 or CD21
have defect in antibody secretion, germinal center formation, and affinity maturation (57). CD20,
+
which is a mature B-cell marker, functions as a membrane-embedded Ca2 channel. It is
important to note here that, the CD20 monoclonal antibody (ritixumab) is the first FDA approved
monoclonal antibody for clinical use in cancer immunotherapy (follicular lymphoma) (18).
Another component of the multi-protein cell surface BCR complex is the transmembrane
immunoglobulin-αβ heterodimer. Igαβ facilitates the recruitment of Src tyrosine kinases to their
cytoplasmic ITAMs domains, and thus is essential for initiating BCR signaling and B cells
activation (56).
13
Several transcription factors that regulate early stages of B cell development have been
identified. In particular Pax5 transcription factor has been shown to play a critical role for B cell
lineage commitment and differentiation (18). For example, Pax5-defecinet mice have been
shown to have an arrest in B-cell development (59). In addition, a significant number of Pax5
regulated genes (approximately 170 genes) have been shown to play an important role in B-cell
signaling, adhesion, and migration (60). Furthermore, Pax5 has also been implicated in human B
cell malignancies, as several cases of acute lymphoblastic leukemias (ALL) and non-Hodgkin
lymphomas have been shown to harbor somatic PAX5 mutation (chromosomal translocations)
(61).
The B cell response is initiated at the boundary between T and B-cell areas in the spleen
and lymph nodes. Specific antigen recognition by the BCR is the first step in the initiation of B
cell signaling and activation. Interactions between B cells, that have captured and processed
antigen, and activated T cells, which have been primed by follicular DCs, lead to the expansion
of antigen specific B cells and to their differentiation into short-lived plasma cells, which
produce low-affinity antibody of IgM or IgD isotype without somatic mutation (62). The
formation of GC reaction follows this extra-follicular response. Further signals from follicular
helper T cells at the GC result in B-cell proliferation, isotype switch, and affinity maturation of
antigen-specific B cells, leading to the generation of memory B cells and long-lived plasma cells
that produce high affinity somatically mutated antibodies of switched isotypes (IgG, IgA, or IgE)
(49). Germinal center plasma cells then migrate to bone marrow and continually secrete
antibodies, thus maintaining constant levels of protective antibodies. Plasma cell migration to
bone marrow is regulated by the bone marrow stromal cells, which provide the attracting
chemokine CXCL12 and the survival cytokines, such as IL-6 (63).
14
Several B cell subsets with distinct functions have been identified. B-1 and marginal zone
+
(MZ) B cell subsets have been described in murine models. Murine B-1 cells are a CD5 B cell
subpopulation which differ from conventional B cells (B-2) by their phenotype, localization, and
+
-
self-renewing capacity (18). B-1 B cells are further divided into B-1a (CD5 ) and B-1b (CD5 )
cell subsets (64). B-1a subset functions to provide IgM antibodies during innate immune
responses against bacterial infection. While, B-1b cells subset provide long-term adaptive
antibody responses during bacterial infections (65). MZ B-cells, which are located in the
periarteriolar lymphoid sheath of the murine spleen, function as the first line of defense against
blood-born encapsulated bacteria (66). In addition to B-1 and MZ B cells, several subpopulations
of B cells in peripheral blood have also been characterized according to specific surface marker
+
-
-
+
+
-
+
+
+
expression, such as IgM IgD CD27 (immature), IgM IgD CD27 (naïve), IgM IgD CD27
-
-
+
(marginal zone, unswitched memory), IgM IgD CD27 (germinal center, switched memory),
high
CD38
IgM
high
(activated), and CD38
high
-
IgM (plasmablast) (16). More recently, a subset of
rare antigen-specific regulatory B cells (B10) with a unique phenotype
high
(CD1d
+
CD5 CD19
high
) has been identified in mice spleens (67). B10 cells have been shown
to play critical regulatory role in the immune system via IL-10 production.
1.3
Regulation of adaptive immunity by the innate immune system:
The cells of the adaptive immune system cannot typically recognize most antigens by
themselves; their responses are usually modulated by innate immune cell PRR-induced signals.
Therefore, innate and adaptive immunity work together to effectively target the adaptive immune
15
responses toward pathogens and allow the adaptive immune system to distinguish self from
nonself. Triggering the activation and maturation of DCs via recognition of PAMPs by PRRs is
the most widely studied mechanism that bridges innate and adaptive immunity (68). Recent
advances in the field of innate immunity however, have identified critical roles for other innate
immune PRRs, as well as other innate immune cells, in orchestrating the function of both innate
and adaptive immune responses. Here I will provide background about the role of various innate
immune cells in adaptive immune cell regulation, cross-talk between innate and adaptive
immune systems, and the impact of various innate immune receptors signaling pathways on
adaptive immune cell responses.
1.3.1 Innate immune cell-dependent regulation of the adaptive immune system:
The expression and production of cytokines upon recognition of PAMPs or endogenous
danger signals by cells of the innate immune system, including monocytes/macrophages, DCs,
NK cell, NKT cells, and neutrophils, play a critical role not only in innate immunity but also in
immune regulation of the adaptive immune system (2). DCs are the major antigen-presenting
cells of the immune system that have an important role in the induction and regulation of
immune responses against pathogens (69). Through expression of several PRRs such as TLRs
and C-type lectins, DCs detect and initiate innate and adaptive immune responses that lead to
pathogen elimination and/or control. In addition, DCs provide stimulatory signals and interacting
with several cells of the innate and adaptive immune systems, such as NK-, NKT-, T-, and Bcells (70). Activated cDCs produce several cytokines that are crucial for promoting cytotoxic T
lymphocyte (CTL) response (via IL-12) and enhancing NK cell activities and survival (via IL-12,
IL-15, and IL-18) (71). In the absence of inflammatory stimuli, DCs are in an immature stage
16
and induce tolerogenic T cell responses (72). However, in an inflammatory microenvironment,
such as in the presence of TLR ligands and inflammatory cytokines and chemokines, DCs have
an enhanced ability to capture antigens, mature, and migrate to lymph nodes, where antigenspecific adaptive immune responses are induced (72). DCsꞌ maturational processes involve
expression of MHC class II, co-stimulatory molecules, such as CD80 and CD86, lymph node
homing receptors, such as CCR7 and CD62L, and production of pro-inflammatory cytokines and
+
+
chemokines, such as IL-12, type I IFNs, and TNFα (73). DCs can activate CD4 and CD8 T
+
+
cell responses either by direct antigen-presentation to CD4 or CD8 T cells or by cross+
presentation to CD8 T cells (73, 74). Moreover, it has been proposed that, in addition to their
immunostimulatory function, DCs have an immunoregulatory function leading to suppression of
T cells responses and control of excessive inflammatory reaction (75).
NK cells have also been shown to play critical roles in shaping adaptive immunity.
Several reports have demonstrated that, in addition to their function in the innate immune
response, NK cells play an important role during the induction of adaptive immune responses.
For example, IFNγ production from NK cells have been shown to enhance cDCs maturation and
promote the differentiation of DCs that are capable of inducing efficient CTL responses (76).
In addition, NK cells were shown to bridge innate and adaptive immune responses by providing
signals for augmenting Th-1 immune responses by production of IFNγ in response to
inflammatory stimuli (77, 78) and inducing tumor-specific CTLs (79). In addition, it has been
shown recently that NK cell-mediated cytotoxicity of antigen-expressing target cells triggers
DCs cross-presentation and thus, facilitates induction of robust antigen-specific adaptive
immune responses (80).
17
NK cells have also been shown to play critical role for B cell activation and the
promotion of isotype class switching. For example, B cell activation by NK cells has been shown
to trigger the production of antigen-specific antibodies with IgG2a and IgG1class-switch isotype
(81). NK cell depletion, via administration of PK136 anti-NK1.1 antibody, has been shown to
affect both the number and the maturation state of DCs in the lymph node (82). Moreover,
depletion of NK cells has also been shown to inhibit the generation of anti-tumor T-cell
responses (79). Interestingly, NK cells have also been shown to have inhibitory roles during
adaptive immune responses. For example, depletion of NK cells after murine cytomegalovirus
(MCMV) infection has been shown to result in enhanced proliferation of CD8+ T cells and
+
+
increased production of IFNγ by both CD4 and CD8 T cells (83). Additionally, depletion of
NK cells have been shown to enhance the tumor-specific CTL responses to MHC class I positive
lymphoma (84), demonstrating that NK cells have both immuno-stimulatory and immunoinhibitory roles during adaptive immune responses. It is important to note here that depletion of
NK cells by NK1.1 antibody can deplete other cells that express the NK1.1 molecule, such as
NKT cells, thus a role for other cells expressing the NK1.1 cannot be excluded in these types of
experiments. Indeed, several reports have demonstrated that enhancing the activation of NKT
cells can also positively influence the initial activation of DCs and/or NK cells, thereby
increasing DC-dependent anti-tumor and anti-viral adaptive immune responses (85-89).
In addition to DCs and NK cells, neutrophils have also been described to play critical role
in bridging innate and adaptive immune responses (90). Neutrophils have been shown to produce
several cytokines and chemokines (such as, IL-1α, IL-1β, IL-6, IL-10, TGFβ, IL-12, IFNα, IFNγ,
G-CSF, and GM-CSF) that are crucial for regulating innate and adaptive immunity (90). For
example, neutrophil derived cytokines have been shown to induce DC maturation and to enhance
18
IL-12 and TNFα production from DCs both in vitro and in vivo (91). Neutrophils have also been
found to directly stimulate the production of IFNγ by human NK cells, thus influencing DC
maturation and Th-1 immune responses (92). Furthermore, human neutrophils have been shown
to directly interact with B- and T-cells to influence their functions (93). Human neutrophilsderived cytokines, BAFF and APRIL, have been shown to be crucial for the survival, maturation
and differentiation of B lymphocytes (94). In addition, the neutrophil-derived chemokines CCL2,
+
CCL20, CXCL9, and CXCL10 have been shown to attract CD4 Th-1 and Th-17 cells to sites of
inflammation (95). Furthermore, human and mouse neutrophils have been shown to crosspresent exogenous antigens, as immunization of mice with OVA-pulsed neutrophils promoted
+
the differentiation of naive CD8 T cells into antigen-specific cytotoxic T cells (96).
A critical component of the innate immune system, the complement system, not only
plays a central role in innate immunity, but it has also been described to play critical roles in
adaptive immune cell responses. Complement, in particular C3, has been shown to modulate Band T-cells responses (97). For example, administration of monoclonal or poly-clonal antibodies
to the C3 receptor, complement receptor 2 (CR2), on human B cells, resulted in B cell
proliferation and differentiation (98). Notably, CR2 is expressed on several innate and adaptive
immune cells including mature B cells, follicular DCs, thymocytes, and sub-population of T cells
(98), implicating a role for C3 in the function of these immune cells. Opsonisation of several
antigens with C3d has been shown to increase their immunogenicity and to lower the threshold
of B cell response to the coated antigen by 1000-fold (99). Mice deficient in CR2, CR1/2 KO
mice, have abnormal B cell survival and impaired B cell functions including reduced levels of
natural antibodies, smaller germinal centers, and irregular humoral immune responses to antigens
(100). Similarly, work in our lab and others have also demonstrated a critical role for CR2 in B
19
cells responses. We have demonstrated that CR1/2 KO mice exhibited impaired B cell responses
following adenovirus administration (101).
The human decay-accelerating factor (DAF), a natural complement system inhibitor, was
shown to retain anti-complement activity when displayed from the surface of the Ad capsid in a
retro-oriented fashion (102). Studies in our lab have shown that mice injected with the “DAFdisplaying” Ad5 vectors, demonstrated significant reductions in pro-inflammatory cytokine
release, reduced endothelial cell activation, minimized activation of pro-inflammatory genes
expression, and reduced plasma ALT levels in mice as compared to unmodified Ad5 vectors.
Also, these results correlated positively with a significantly decreased activation of dendritic
cells, NK cells and T cells (102, 103). Importantly, this modulation of the complement dependent
arm of the innate immune response resulted in significantly reduced induction of Ad neutralizing
antibody responses, as well as in blunted T cell responses to the transgene (HIV/Gag) expressed
by the DAF displaying Ad (103).
The role of complement in T cell responses has also been demonstrated. Several reports
have demonstrated that DCs express a number of receptors specific for complement proteins
(CR3, CR4, C3aR, and C5aR) and ligand binding to these receptors can affect DC maturation
and migration. For example, interaction of C5a with C5aR on DCs, has been shown to induce
DCs maturation and expression of the lymph node homing chemokine receptor CCR7,
implicating an indirect role for C5a-C5aR interaction in T cell activation (104). Similarly,
binding of C3a to C3aR on DCs has been shown to result in IL-12 production, which supports
Th1 immune responses (105). In addition, C3aR KO mice have been shown to have increased
production of the Th2 immune response cytokines IL-4, IL-5, and IL-10 (105), suggesting a role
for C3a-C3aR interaction in mediating Th1 immune responses to antigens. Complements have
20
also been shown to have immuno-inhibitory roles. For example, iC3b has been shown to inhibit
DCs maturation (inhibit expression of CD40 and CD86) and to inhibit production of the proinflammatory cytokines IL-1β, TNFα, and IL12 by DCs (106).
1.3.2 The impact of innate immune recognition on adaptive immune cell responses:
Several PRRs act as signaling receptors to induce production of innate effector molecules
upon pathogen encounter, or when host-derived markers of stress and/ or damage, are present.
These signaling receptors are divided into several classes including TLRs, RLRs, and NLRs
(107). Recognition of PAMPs by TLRs results in activation of NFκB/ AP-1 signaling pathways,
leading to production of pro-inflammatory cytokines and chemokines (such as IL-6, IL-12, and
TNFα) that coordinate innate immunity and initiate adaptive immune responses to various
pathogens (8). In addition, TLRs 3, 4, 7, 8, and 9 can activate the IRF3 and/ or IRF7 signaling
pathways in pDCs during viral infection and result in the induction of multiple genes involved in
innate and adaptive immunity, including type I IFNs (IFNα and IFNβ) (8). Induction of type I
IFNs have been shown to result in activation of NK cells (increased IFNγ production and
cytotoxicity), enhancing DC maturation (increased, IL-12 production, expression of MHC-II, and
costimulatory molecules), and induction of Th1 immune responses (108). In addition, type I
IFNs can directly activate adaptive immune cells. For example, type I IFN receptor signaling in
CD8+ T cells was found to be critical for the generation of effector and memory CD8+ T cells in
response to viral infection (109). Innate immune signaling has also been shown to suppress the
adaptive immune system. For example, TLR2 signaling in DCs has been shown to produce high
levels of IL-10 and little IL-12, thus promoting the differentiation of Th2 cells (which activate
humoral immunity) or regulatory T cells (which suppress cellular immunity) (110).
21
In addition to TLRs, members of the C-type lectin family of cell surface PRRs, called
Dectin-1 and 2, have also been shown to regulate adaptive immunity. Dectin-1 recognizes βglucans from fungal pathogens, such as Candida albicans (111). Dectin-1 stimulation has been
shown to induce DCs maturation and the production of Th-17 supporting cytokine, IL-23 (112).
In addition, when used as an adjuvant, Dectin-1-activated DCs have been shown to favor the
differentiation of T cells into the Th17 phenotype, which is required to clear fungal infection
(112).
In addition to transmembrane receptors on the cell surface and in endosomal
compartments, several studies have shown that intracellular (cytosolic) PRRs, such as RLRs and
some NLRs, can also regulate adaptive immune responses (113). Innate immune sensing of
peptidoglycan by NOD1 and NOD2 proteins has been shown to contribute significantly to the
initiation of the adaptive immune responses. For example, NOD1 stimulation alone has been
shown to drive antigen-specific immune responses toward Th2 phenotype (114). In addition,
antigen-specific T- and B-cell immune responses were severely diminished in NOD1 KO mice
following vaccination (114). In contrast, NOD2 has been shown to mediate the adjuvant effect of
muramyl peptide (MDP) and regulate bacterial immunity within the intestine (115).
The NLR NALP3 inflammasome has also been shown to play a role in regulating
adaptive immune responses. Several adjuvants such as aluminum hydroxide (alum), infections
such as influenza, or products of cell death such as extracellular ATP and uric acid have been
shown to activate the NALP3 inflammasome (116). Activation of the NALP3 inflammasome in
DCs has been shown to activate caspase-3, trigger IL-1β release, and induce anti-tumor adaptive
immune responses (117).
22
The RLRs RIG-I and MDA5 have also been shown to play critical role in antiviral
immune responses. Recognition of viral RNA by the helicase domain of RIG-I and MDA5 leads
to the activation of NFκB and IRF3, thus trigger transcription and production of proinflammatory
cytokines and chemokines, type I IFNs, and induction of Th1 adaptive immune responses (118).
+
A cytosolic DNA sensor has also been shown to enhance cytotoxic CD8 T cell and antibody
responses following DNA vaccination in a TANK-binding kinase 1 (TBK-1) and type I IFNdependent manner (119).
1.4
Vaccine Adjuvants:
Vaccine adjuvants are represented by different classes of compounds including, microbial
products, mineral salts, emulsions, nucleic acids, and liposomes (120). Vaccine adjuvants were
traditionally used to enhance the immunogenicity of vaccine antigens. Adjuvants have been
found to increase the speed of initial response (jump-start innate immunity), increase the
generation of immunological memory, alter the breadth of the response, and provide specific
+
+
types of immune responses, such as Th1 versus Th2 or CD8 versus CD4 T cell responses
(120). The cellular and molecular mechanisms of actions of adjuvants are poorly understood
(120). Vaccine adjuvants can be classified into two major groups, TLR-dependent and TLRindependent (121). TLR-dependent adjuvants act directly on DCs and results in up-regulation of
MHC class II, increased expression of costimulatory molecules, production of pro-inflammatory
cytokines and chemokines, and enhanced migration of DCs to T cell area in lymph nodes (122).
Examples of TLR-dependents adjuvants that are currently in preclinical or clinical studies
include, the synthetic analogs of dsRNA, such as Poly I:C (TLR3) (123), monophosphoryle lipid
A (MPL) (TLR4, currently licensed for HBV and papilloma vaccines) (124), bacterial flagelline
23
(TLR5) (125), Imiquimod (TLR7) and Resiquimod (TLR7-TLR8) (126), and CpG-ODN
(TLR9) (127).
The TLR-independent adjuvants include alum and the squalene-based oil-in water
emulsions MF59 and AS03. These latter adjuvants are widely used in human vaccines (128).
While the molecular mechanism of action and the target cells of these adjuvants are still
unknown, several studies have found that these adjuvants enhance antigen up-take by APCs
(129). However, recent studies suggested that, in addition to antigen delivery, these adjuvants
might have immunostimulatory functions in vivo. For example, Alum has been found to activate
the NALP3 inflammasome to produce mature IL-1β and to trigger necrotic cell death and the
release of the endogenous danger signal, uric acid (120). In addition, MF59 has been found to
stimulate the release of lymphocyte chemoattractants, such as CCL2, CCL3, CCL4, and CXCL8,
from human macrophages, monocytes, and granulocytes in vitro (130). In addition, MF59 has
also been shown to enhance monocyte differentiation into DCs in vitro (130). Similar to alum,
when administered in vivo, MF59 has been found to trigger a local immunostimulatory
environment, an environment that resulted in differentiation of monocytes into inflammatory
DCs expressing high levels of MHC class II molecules.
1.5
The immuno-modulatory molecule: recombinant Eimeria Tenella derived Antigen
(rEA)
The bovine small intestine is resistant to tumor formation, and an activity isolated from
that small intestine was isolated and identified as a protein derived from Eimeria tenella. A
recombinant form of this Eimeria tenella derived antigen (rEA) has been generated. (131). The
EA originates from an endemic pathogenic protozoan of the Eimeria genus Apicomplexa phylum
24
that include Toxoplasma, Plasmodium, Eimeria and cyclospora genera. rEA has been shown to
induce IL-12 release from mouse DC in vitro and to trigger potent production of IL-12 and other
proinflammatory cytokines and chemokines in vivo (131, 132). rEA has also been shown to
trigger potent IFNγ release from NK cells and to enhance NK cytolytic activity toward S-180
tumor cells both in vitro and in vivo, an activity that was completely dependent on IL-12 release
from DCs (131). Studies in our lab have also shown that NK and NKT cell activation was
significantly induced following systemic rEA administration (133). The enhanced activation of
DCs and NK cells as well as the increased production of proinflammatory cytokines and
chemokines by rEA positively correlated with enhanced humoral and Th1 cellular immune
responses to the co-administered Toxoplasma gondii antigen, resulting in protection against
Toxoplasma gondii infection in mice (134). rEA has also been shown to be an efficient
immunomodulator, having both anti-viral and anti-tumor properties in mice (131, 135, 136).
Moreover, rEA showed no evidence of toxicity in pre-clinical (131) and clinical trials (137).
Specifically, in a phase I human clinical trials, rEA was used as a single agent in advanced
gynecological cancer patient. The results from the trial showed a reduction in CA125 levels as
indication of treatment-associated efficacy and importantly, no severe adverse reactions were
reported in human clinical trials despite detection of increased IL-12p70 responses in up to 30%
of the treated cancer patients (137).
The rEA protein has a relatively high amino acid sequence homology (67%) and shares
very similar biological activities in vitro and in vivo with T. gondii-derived profilin-like protein,
both of which trigger potent IL-12 responses in DCs. The profilin induced responses were
completely dependent upon the adaptor protein MyD88 and at least partially mediated via
TLR11 (138). Moreover, it has been shown in vitro that human TLRs (TLR2, TLR3, TLR4,
25
TLR5, TLR7, TLR8 and TLR9) do not transduce rEA signaling (135). Therefore, TLR11 has
been suggested as the rEA receptor mediating rEA signaling, but this notion remains to be
confirmed. Since no functional human TLR11 homolog has been discovered, these facts leave
unidentified the mechanism underlying rEA action in humans, and opens a discussion regarding
other PRRs that may be involved in rEA signaling (135). Additionally, it is not known what cell
types are primarily responsible for mediating rEA-triggered responses in vivo. Studies in our lab
have shown that the magnitude and quality of HIV-Gag-specific T cell responses were
significantly improved when rEA expressing Ads and antigen expressing Ads were administered
together (139). In addition, inclusion of rEA in the Ad based vaccination regimen improved the
+
in vivo cytolytic activity of the HIV-Gag-specific CD8 T cells (102).
1.6
Harnessing innate immunity by the SLAM family of receptors adaptor EAT-2
1.6.1 The signaling lymphocytic activation molecules (SLAM) family of receptors
The SLAM family of receptors is a group of type I transmembrane receptors that play an
important role in immune regulation (140). These receptors belong to the CD2 superfamily of
immunoglobulin (Ig) domain-containing molecules, are expressed on the surface of wide variety
of immune cells, and are not found in non-immune cells. SLAM family of receptors includes
SLAM (CD150; SLAMF1), 2B4 (CD244; SLAMF4), Ly-9 (CD229; SLAMF3), natural killer, Tand B-cell antigen (NTB-A) or Ly108 (in the mouse) (SLAMF6), CD84 (SLAMF5), and CD2like receptor activating cytotoxic cells (CRACC; CD319; and SLAMF7) (Table 1) (141).
26
Table 1: Expression pattern of SLAM family of receptors in hemopoietic cells
Table 1
Receptors
Physiological
ligand
SLAM
SLAM
2B4
NTBA/L108
CD48
NTBa/Ly108
Ly-9
Ly-9
CD84
CD84
CRACC
CRACC
Cellular distribution
T, B, DCs, Platelets,
and Macrophages
NK cells, CD8+ T cells,
DCs, and Macrophages
NK, DCs T, and B cells
NK, DC, neutrophils, B,
and T cells
T, B, NK, DCs,
Platelets, and
Macrophages
T, B, NK, DCs, and
Macrophages
Associates with
SAP, EAT-2, ERT, SHP2, SHIP1,
and FYN
SAP, EAT-2, ERT, SHP2, SHP2,
and LAT
SAP, EAT-2, ERT, SHP2, SHP2,
and LAT
SAP, EAT-2, ERT, SHP2, and AP2
SAP, EAT-2, ERT, SHP2, and
SHIP1
EAT-2, SHP1, SHP2, and SHIP1
Structurally, SLAM family of receptors contain an extracellular domain composed of two
immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic domain that
carry one or more copies of a unique intracellular tyrosine-based switch motif (ITSM) (141). The
SLAM family of receptor ITSM motif has a high affinity for a family of adaptor molecules
called SLAM-associated protein (SAP) family of adaptors. The SLAM receptors have a unique
feature compared to most other receptors expressed in immune cells, in that they are self-ligands.
One exception is 2B4, which interacts with CD48, a member of the Ig superfamily that is
expressed in most hematopoietic cells (142). Thus, SLAM family of receptors can be triggered
by homotypic or heterotypic cell-cell interactions through their respective extracellular domains.
Several lines of experimental investigation have been utilized to identify the role of these
receptors in immune cells including, antibody stimulation approaches, ectopic expression of
ligands on target cells, genetic linkage analysis, and analysis of genetically modified mice (140).
Recent data suggest that these immuno-modulatory receptors perform multiple functions in
27
+
hematopoietic cells, including roles in regulating cellular costimulation, NK- and CD8 T-cellmediated cytotoxicity; macrophage, DCs, T cell and NK cell cytokine production; adhesion
between hematopoietic cells; the development of innate T lymphocytes; as well as regulating
functions of neutrophils and macrophages (143, 144).
The SLAM family of receptors genes are located within a 400 kilobase (kb) fragment on
chromosome 1 in both humans and mice (145). The similarities between SLAM family genes in
sequence, genomic organization, gene localization, and ITSMs, imply that the SLAM family was
generated by sequential duplication of a single ancestral gene. Several reports demonstrated the
presence of multiple splice forms and polymorphisms for many of the genes encoding the SLAM
family members. For example, polymorphisms of the Ly108-encoding gene, in mice, were found
to correlate with susceptibility to the autoimmune disease systemic lupus erythematosus (SLE)
(146). Furthermore, polymorphisms in the Ly-9 and 2B4-encoding genes were respectively
linked to susceptibility to SLE and rheumatoid arthritis (RA) in humans (147, 148).
1.6.2 Signal transduction of SLAM family of receptors
SLAM family of receptor initiated intracellular signaling is mediated primarily by the
SAP family of adaptors. The SAP family of adaptors are composed of three members named,
SLAM-associated protein (SAP), Ewing's sarcoma-associated transcript-2 (EAT-2), and EAT-2related transducer (ERT) (Table 2) (149).
Table 2: The SAP family of SH-2 domain containing adaptors:
The SAP family of adaptors are mainly expressed in immune cells, with SAP present in T cells,
NK cells, NKT cells, and some B cells. EAT-2 is found in NK cells, DCs, and macrophages,
28
while ERT is expressed only in mouse NK cells. The SAP encoding gene is located in the X
chromosome in humans and mice while EAT-2 and ERT are located on human chromosome 1.
The ERT encoding gene is a pseudogene, being nonfunctional in humans.
Table 2: The SAP family of SH-2 domain containing adaptors:
Table 2
Chromosomal
location
X (human and
SAP (SH2D1A)
mice)
1 (human and
EAT-2 (SH2D1B1) mice)
1 (mice and
pseudogene in
ERT (SH2D1B2)
human)
Adaptor
Expression
pattern
T, B, NK, NKT,
and platelets
NK, DCs, and
macrophages
Signaling
partners
FynT, PIX, and
Nck (arginine 78)
? PLCγ (Cterminal tyrosine)
NK cells
? (C-terminal
tyrosine)
Human
disease
XLP
none
none
These intracellular adaptor molecules are composed primarily of a Src-homology 2 (SH2) domain, in addition to a short carboxy-terminal region. Through their SH-2 domain, SAP
adaptors associate with the tyrosine-based switch motif TI/VYxxV/I in the cytoplasmic domain
of SLAM receptors with high affinity and specificity (141). All SLAM receptors can interact
with either of the adaptors, except CRACC which interacts only with EAT-2 (150, 151).
The importance of the SAP adaptors and SLAM receptors was indicated in 1998 after
the discovery that SAP is mutated and inactivated in 60%-70% of cases of a human
immunodeficiency, X-linked lymphoproliferative (XLP) disease (152). XLP patients have a
number of distinguishing features characterized by ineffective responses to Epstein-Barr virus
(EBV) infections and a higher frequency for development of malignant lymphomas (152). This
is correlated with defects in the development and function of several immune cell types,
+
+
including CD4 T-, CD8 T-, NK-, NKT-, and B-cells (140). Similar to SAP-deficient humans,
mice lacking one or more members of SAP family adaptors have a similar phenotype (153). SAP
29
+
deficient mice have defects in CD4 T cells function, including Th2 cytokine production and
defective TFH cell function, which impact upon germinal center formation and antibody
+
production by B cells (154). In addition, SAP deficient mice have reduced CD8 T- and NKcells cytotoxic capabilities, absence of NKT cells, and reduced IFNγ production by NK cell
(155). Mutation of EAT-2 or ERT was not described in XLP disease, however, mice lacking
EAT-2 or ERT have been shown to have altered NK cells functionality (156). In addition, work
by the Veillette et al group showed that mice lacking all SAP family of adaptors have severe NK
cells defects as compared to mice lacking individual SAP family adaptors (157).
SAP and EAT-2 adaptors have been shown initially to prevent the binding of inhibitory
signaling molecules to the SLAM family of receptors ITSMs (158). These molecules include, the
SH-2 domain-containing phosphatases (SHP)-2 and (SHP)-1, Csk, and SH-2 domain-containing
5’ inositol phosphatase (SHIP)-1 (153). This led to the conclusion that SAP adaptors are natural
blockers that allow SLAM family of receptors to mediate positive immune cell activation.
However, subsequent studies showed that SAP, EAT-2, and ERT harbor specific sequences that
allow them to couple SLAM family of receptors to active biochemical signaling molecules (140).
For example, the SAP adaptor has been shown to have a specific sequence within the SH-2
domain, arginine 78 (R78) motif which associates directly with the protein tyrosine kinase FynT
(159). This association links SLAM family of receptors to protein tyrosine phosphorylation
signals (160, 161). In addition, activation of SLAM (CD150) receptor signaling in B and T cells
by antibody crosslinking experiments resulted in PI3K activation and enhanced Akt
+
phosphorylation (162, 163). Furthermore, SLAM-SAP signaling in CD4 T cells prolongs
30
protein kinase C theta (PKCθ) recruitment to the site of contact with antigen presenting cell and
resulted in increased NFκB activation (164).
EAT-2 and ERT do not contain the arginine (R87) motif that is found in the SAP adaptor,
however, similar to SAP, EAT-2 and ERT have been shown to transduce positive signals
downstream of SLAM family of receptors in human and mouse NK cells (144, 150). The
activation function of EAT-2 and ERT was dependent on phosphorylation of tyrosine residues
directly located in their short carboxyl-terminal tails (165). Mouse EAT-2 and ERT contain two
tyrosine residues (tyrosine 120 and 127) in their short C-terminal tail (156). In contrast, the
human EAT-2 adaptor contains only one tyrosine residue (tyrosine 127) in its C-terminal tail
(144). Phosphorylation of EAT-2 and ERT tyrosine residues serves as a docking site for other,
yet unidentified, SH-2 domain-containing downstream effector molecules (166). Recent data
suggest that phospholipase C (PLC)-γ is recruited to tyrosine 127 in human EAT-2 (167).
Furthermore, over-expression and binding studies indicate that EAT-2 directly binds to the
catalytic domain of the Src family kinases, Fyn, Hck, Lyn, Lck, and Fgr (168). It is important to
note here that, the role of the EAT-2 adaptor in immune cell function has been studied primarily
in human and mouse NK cells. Whether EAT-2 or ERT harbor critical function in immune cell
types other than NK cells, still needs to be clarified.
1.7.
Adenoviruses as vaccine vectors:
Replication-deficient adenovirus based vectors (Ads) have been the focus of considerable
interest in the last few years for their potential applications in both gene therapy and vaccine
developments (169). Adenoviruses are a family of non-enveloped viruses containing an
icosahederal protein capsid with a 30- to 40-kb linear double-stranded DNA genome. In general,
31
of the immunologically distinct human Ad serotypes, none are associated with any neoplastic
disease, with most causing relatively mild, self-limiting respiratory illnesses in immunocompetent individuals (170). At least 51 serotypes of human Ad have been identified, which are
categorized into six subgroups (A-F), primarily based upon different red blood cell agglutinating
capabilities of the various subgroups. Ad serotypes 5 (Ad5) and Ad2, both belonging to subclass
C, are the most extensively studied and characterized both relative to general Ad biology, as well
in regard to utilization as a gene transfer vector.
Several generations of recombinant Ad vector has been generated. First generation
adenovirus vectors are constructed such that a transgene replaces only the E1 region of genes
([E1-] Ad) or E1 and portion of E3 ([E1-, E3-] Ad); thus, 90% of the wild-type Ad genome is
retained in the vector. Ad vectors can be propagated in human cells (HEK 293 cells) engineered
to express the E1 proteins in trans (171, 172). Ad vectors with additional deletions in their
genome in the E2A, E2B and E4 Ad genes have been generated (accommodated by use of newer
generation, trans-complementing packaging cell lines) (173-176). These advanced generation Ad
vectors produce fewer Ad derived gene products as compared to first generation Ads, and can
minimize the induction of vector-specific adaptive immune responses (174, 177).
Ad vectors posses several important advantages, the most important of which is that they
can be easily and routinely produced to high titers in a good manufacturing practice (GMP)
13
compliant fashion (up to 1×10
vp/ml). Additionally, Ad vectors allow for efficient transduction
of various proliferating and quiescent cell types, they can allow for transfer of large segments of
foreign DNA (up to 35 kB in some systems), and most importantly, Ad vectors do not integrate,
and therefore are much less likely to cause insertional mutagenesis or germ line transmission
32
associated problems, in contrast to integrating virus based vectors, such as retrovirus and
lentivirus based gene transfer systems (178).
Ad vectors rapidly activate innate immune responses as well induces potent cellular and
humoral adaptive immune responses, against both the vector and transgene product being
expressed (179-181). Upon initial introduction into a host, the innate immune response can be
initiated following the binding or coating of the Ad vector capsid with several (extra-cellular)
factors including: surfactant-A (SP-A), lactoferrin, pre-existing immunoglobulin, and protein
members of the complement pathways, both classical (C1q, C4) and alternative (Factor B, Factor
D) (182, 183). Administration of Ad vectors results in the immediate production (1-6 hours post
injection) of various pro-inflammatory cytokines and chemokines, as well as type I IFNs in mice,
non-human and human primates (179, 181, 184-186). Specifically, high dose, intravascular
administrations of Ad vectors have been found to induce high levels of the cytokines IL-1α, IL1β, TNFα, IL-6, IL-12, and IFNγ and the chemokines RANTES, MCP-1, KC, MIP-1α, MIP-1β,
and IP-10 (133, 181, 184, 187). The origins of these pro-inflammatory mediators in vivo is not
fully known but is likely from multiple sources inclusive of Kupffer cells, macrophages,
endothelial cells, as well as Ad transduced tissues and organs themselves (184, 188).
Furthermore, Ad vectors can either activate of directly transduce various immune cells types in
the liver and spleen including: dendritic cells (189), both pDCs (185) and cDCs (190),
macrophages (191), and to a lesser extent NK cells (191, 192). The induction of type I IFNs is
critical for innate immune defense against Ad vectors in vivo (193); the maturation of DCs (194);
and the regulation of the induction of pro-inflammatory cytokines (195, 196).
33
1.7.1 Molecular basis for cellular recognition of Adenovirus vectors
Ad5 vectors interact with both the Coxsackie-Adenovirus receptor (CAR) (via the Ad
fiber knob domain) and with cellular integrins (via the Ad penton base RGD motifs) to initiate
host cell penetration (197, 198). The penetration process itself will also simultaneously trigger
cellular pro-inflammatory innate immune responses (199). After internalization and upon
endosomal escape, Ad vectors have been shown to activate MAPK and NFκB signaling
pathways via both TLR dependent and TLR non- dependent mechanisms (184, 195). We and
others have shown that, Ad vectors activate TLR (TLR2, TLR4, and TLR9) signaling and induce
various cytokines and chemokines responses (181, 184, 185).
In addition to TLRs, significant evidence has been accumulated in recent years
implicating a TLR9-independent mechanism for sensing Ad5 double-stranded DNA (dsDNA)
genomes (195, 200, 201). Takaoka et al. showed that a dsDNA sensor called DNA-dependent
activator of interferon regulatory factors (DAI) activates type I interferon in response to DNA
viruses in L-929 cells, but subsequent studies suggested the presence of other cytoplasmic DNA
sensor(s) (119, 202, 203). Ads have also been shown to activate the NALP3 inflammasome. The
activation of the NALP3 inflammasome by Ad derived dsDNA leads to caspase-dependent
activation of IL-1β and induction of pro-inflammatory cytokine and chemokine responses
including elevations of IL-6, MIP-1β, IP-10, and MCP-1 (204). In addition, Ad mediated
disruption of lysosomal membranes, and the release of cathepsin B into the cytoplasm, are
required for Ad-induced NLRP3 inflammasome activation (205). Furthermore, Ad5 activation of
NLRP3 also induced necrotic cell death, resulting in the release of the proinflammatory molecule
HMGB1 (High-mobility group box 1 protein), a recently identified DAMP that mediates the
response to infection, injury, and inflammation (205, 206). In addition, it has been demonstrated
34
that adenovirus virus-associated RNA (VA) , a dsRNA that inhibits interferon and RNAi sensing
mechanisms, is no less recognized by RIG-I, a cytosolic pattern recognition receptor, and
activates RIG-I downstream signaling, leading to the induction of type I IFNs (207).
1.7.2 Adaptive immune responses to Ad vector expressed transgenes
Ad-based vectors have a potent ability to induce potent humoral, but more importantly,
cellular (CTL) immune responses to expressed foreign antigens, and have therefore recently
received much attention for use in a number of vaccine based applications (169, 208).
Specifically, E1 deleted Ad5 vectors expressing the HIV-1 gag, pol and nef genes have been
utilized in human trial subjects (209, 210). The results from the early-phase clinical trials
demonstrated that the Ad5 vector-based vaccines elicited some of the most potent, HIV-1specific cellular immune responses in humans to date, however, the presence of pre-existing
Ad5-specific neutralizing antibodies partially suppressed these responses (209, 210).
Utilization of advanced generation Ad vectors have also recently been shown to allow for
improved efficacy in several vaccine based applications (211-213). Specifically, [E1-,E2b-]Ad5
vectors were able to induce heightened antigen-specific T cell responses in mice and primates
despite pre-existing Ad5 immunity (211, 212, 214). In contrast to E1 deleted Ad vectors, these
types of Ad vaccines can show strong efficacy despite the existence of pre-existing Ad+
immunity, possibly by their avoidance of CD8 T cell responses directed to the Ad polymerase
gene (211-215). [E1-, E2b-]Ad vectors, expressing tumor derived antigens were also able to
induce beneficial, cytolytic T cell responses that promoted tumor regression in murine models
(213). Based upon these improvements, a phase I/II clinical trial is currently underway, utilizing
a CEA (Carcinoembryonic Antigen) expressing [E1-,E2b-]Ad5 vector in an attempt to safely
35
induce beneficial, CEA specific adaptive immune responses in patients (both Ad5 naive and Ad5
immune) bearing CEA expressing tumors.
In summary, use of Adenovirus vectors for vaccine development is a promising approach
in terms of inducing potent adaptive (cellular) immune responses to vaccine antigens. Harnessing
the innate immune system is a valid strategy for shaping the adaptive immune responses to
vaccine antigens. Several classes of vaccine adjuvants are currently under evaluation in both preclinical and clinical trials. However, their mechanisms of actions are not completely understood.
Understanding the molecular mechanisms of vaccine adjuvants and further developments of Ad5
vaccine vectors to enhance their efficacy are now justified. Possibly a combination of Ad5 vector
adjuvant activity and activation of a unique arm of the innate immune system may result in
production of more efficacious Ad-based vaccine platforms. In this dissertation, we described a
novel class of Ad5-based vaccine vectors that expresses the SLAM family of receptors adaptor
molecule, EAT-2, and outline superior qualities relative to current generation rAd5-based
vaccines. We also proposed to utilize the novel immunomodulatory protein “rEA” to enhance
vaccines efficacy. Potentially in combination with other vaccine approaches, such as vectors- or
DCs-based vaccines, to further enhance induction of antigen specific adaptive immune
responses.
36
Chapter II
Expression of the SLAM family of receptors adapter EAT-2 as a novel strategy for
enhancing beneficial immune responses to vaccine antigens
This chapter is the edited version of a research article that was published in the Journal of
Immunology, Volume 186, Issue 2 (722-732), Jan 15, 2011.
Authors: Aldhamen Y.A., Seregin S.S., Appledorn D.M. , Liu CJ, Schuldt N, Godbehere S, and
Amalfitano A.
37
2.1 Introduction
Development of an effective vaccine to prevent infections by the human
immunodeficiency virus-1 (HIV-1) is an important goal. Most recently, a human clinical trial
demonstrated that a prophylactic vaccine to HIV may indeed be possible (216). However, the
results of that trial combined with recent results derived from the Merck® sponsored STEP trial,
suggest that a more potent vaccine capable of inducing greater levels of antigen specific adaptive
immune responses may demonstrate greater efficacy to prevent HIV infection (210, 217).
Incorporation of adjuvants into vaccine formulations can improve the induction of antigen
specific adaptive immune responses (218-220). Pro-active induction and/or harnessing of
beneficial innate immune responses may be the mechanism underlying the effectiveness of
certain adjuvants to significantly contribute to the ability of vaccines to generate adaptive
immune responses (2, 108). Here, we wished to evaluate the potential of improving vaccination
efficacy by presenting a target antigen while simultaneously activating novel arms of the
mammalian immune system. While in previous studies we and others have explored the potential
of modifying TLR dependent innate immune responses to facilitate improved efficacy of virus
based vaccines (133, 221), in this study we set out to determine if targeted manipulation of the
signaling lymphocytic activation molecule family of receptors (SLAM) pathway could also
facilitate improved induction of HIV-1-Gag-specific, adaptive immune responses by vaccine
platforms.
In this chapter, I will provide a brief background about human immunodeficiency virus
type I (HIV-1) biology, innate and adaptive immune responses to HIV-1, immune evasion
mechanism of HIV-1, describe the current state of HIV-1-vaccine field and outline strategies for
the development of an effective HIV-1 vaccine, and finally introduce a novel strategy for HIV-1
38
vaccine development by targeting the SLAM family of receptor signaling by EAT-2 adaptor
protein.
2.1.1. Human immunodeficiency virus (HIV) and AIDS global epidemic
HIV-1/ AIDS continue to be a significant health threat both in the United States and
world-wide. AIDS was first recognized in 1981 and by 1983; HIV was identified to be the
causative agent of this devastating disease. To date, more than 20 million people worldwide have
died as a result of HIV-1infection. The global number of people living with HIV-1 was estimated
to be 33.3 million in 2009 (222). HIV infection is associated with high morbidity and mortality
contributing to 2 million deaths and 2.7 million being newly infected each year (222). Despite
the intensive research in the past three decades, the AIDS epidemic is still spreading in an
uncontrolled fashion across the globe, emphasizing the need to urgently develop effective
vaccines, microbicides, and other preventative strategies to blunt the progress of the epidemic
and to stop HIV-1 transmission.
Since its discovery, the origin of HIV virus remains a topic of great interest for both the
public and scientific community. HIV belongs to the lentiviral genus of the family retroviridae.
Phylogenetic analysis of the lentiviral lineage has revealed that HIV was originated from
multiple zoonotic transmissions of simian immunodeficiency virus (SIV) from non-human
primates (NHP) into humans in West and Central Africa (223). More than 40 different
nonhuman primate species harbor SIV infections. Thus, several independent zoonotic
transmission events from NHP to humans have generated several HIV lineages; HIV type 1
(HIV-1) groups M (main group; has nine subtypes), O (outlier group), N (non-M/ non-O), P
(proposed groups), and HIV type 2 (HIV-2) groups A-H (224). Each group of HIV causes a
39
relatively different epidemic. HIV-1 group M is responsible for the global HIV infection
(approximately 33 million infected individuals), group O causes tens of thousands of infections
in West and Central Africa, group N has been found in few people in Cameroon, and group P
was recently identified in two individuals originating from Cameroon (224). HIV-2, on the other
hand, causes a relatively attenuated pathogenicity in human.
2.1.2. HIV-1 genetic diversity and its impact for HIV-1 control
Similar to most RNA viruses, the HIV-1 virus demonstrates an enormous genetic
variability and rapid evolution rate due to the poor fidelity of reverse transcriptase and a lack of
proofreading machinery (225). The estimated mutation rate for HIV-1 is 5 × 10
-5
mutations/site/generation (225). HIV-1 mutation and recombination result in the rapid generation
of genetically diverse viral populations within each infected individual (226). Several full-length
genome sequencing studies have revealed that recombination between strains is a much more
powerful and relevant force in shaping HIV-1 evolutionary patterns than individual point
mutation frequency (227). Further analysis revealed that recombination does not appear to be
limited by sequence similarity, as recombination has occurred between strains from different
HIV-1 groups (groups M and O) as well as between and within group M subtypes (228). As a
result of this rapid evolution and diversification of the HIV-1 virus, the genetic diversity within
the HIV-1 group M envelope, Env, is estimated to be as high as 35% and recombinants are
currently estimated to be responsible for at least 20% of HIV-1 infections worldwide (229). This
issue represents one of the most challenging factors for controlling disease progression, in
designing effective anti-retroviral therapies, and in manufacturing effective therapeutic and
preventative HIV-1 vaccine (230).
40
2.1.3. Immune response to HIV-1 and immune evasion mechanisms
2.1.3.1 Innate immune responses to HIV-1:
It is well established that HIV-1 infection is associated with the modulation and
dysregulation of the immune system (231). Adaptive immune responses occur late in HIV-1
infection, with T cell responses appearing 1-2 weeks after initial infection and neutralizing
antibodies appearing 3 months after infection (232), emphasizing a critical role for the innate
immune system in early anti-HIV-1 responses. The role of the innate immune system in HIV-1
infection has been studied extensively. The innate immune response to HIV-1 includes a variety
of cellular, extracellular, and intracellular components that initiate diverse set of signaling
pathways that restrict growth and replication of HIV-1 (233). The innate immune cells that have
been shown to be involved in protection from HIV-1 infection include: Langerhans cells in
vaginal and foreskin epithelia (234), γδ T cells in rectal and vaginal epithelia, and macrophage,
DCs, and NK cells in the subepithelial tissues (235).
DCs and NK cells have central roles in antiviral immunity by shaping the quality of the
adaptive immune response to viruses and by mediating direct antiviral responses (236). HIV-1
infection has been shown to dysregulate and reduce DCs number in the blood, a phenomenon
that correlates with increased viral load and disease progression (237). The interaction between
HIV-1 and DCs is complex and not yet fully understood. Several DC-expressed surface receptors
(for example, CCR5, DC-SIGN, and CXCR4) have been shown to be target for binding and
internalization of HIV-1 (238). Commonly, recognition of single stranded RNA by TLR-7 in
pDCs, activates IRF signaling pathways that results in, production of type I interferons (as well
as other cytokines), stimulation of cDCs activities, and directly enhancing NK cell responses
(239). HIV-1 infection does not induce cDCs maturation through TLR activation. The
41
mechanism of this inhibition is not fully understood, however, low HIV-1 viral replication within
cDCs, the interactions between HIV-1 and C-type lectins (which abrogate TLR responsiveness),
and prevention of autophagy-mediated viral degradation, are all proposed mechanisms for this
inhibitory process (240). In addition, it has been shown that type I IFN production from pDCs
can facilitate HIV-1 infection by enhancing HIV-1 replication. For example, IFNα production
+
from pDCs has been shown to up-regulate CD4 T cell expression of the pro-apoptotic
molecules, such as TNF-related apoptosis-inducing ligand (TRAIL), death receptor 5 (DR5), and
+
FAS, leading to a progressive CD4 T cell loss (241). Furthermore, it has been shown that HIV1-exposed pDCs can promote the development of Treg cells, which prevent the induction of
effector T cell responses by inhibiting the maturation of cDCs (242). Moreover, as DCs can
regulate NK cell activation, dysregulation of DCs have been shown to contribute to the
dysregulated NK cell function commonly observed in HIV-1 infected individuals.
NK cells prevent the early spread of viruses by producing cytokines and directly killing
infected cells (243). NK cells interact with DCs to shape the magnitude and quality of the
adaptive immune response (244). NK cells responses to viral infection is controlled by several
inhibitory and activating receptors (245). For example, lack of the MHC-I expression (which
binds NK cell inhibitory receptor) on viral infected cells and/ or expression of ligand for NK cell
activation receptors, are two mechanisms that have been described to regulate NK cell responses
during viral infections (243). To date, no specific NK cell receptors that directly recognize HIV1-infected cells have been identified (236). Several lines of investigations have shown that HIV1 virus utilizes several mechanisms in order to evade NK cell responses. For example, it has been
shown that the HIV-1 Nef (negative factor) that selectively down-regulates the expression of
42
HLA-A and HLA-B, but not HLA-C or HLA-E, allows HIV-1 to evade CTL responses,
responses that are largely directed against HLA-A and HLA-B-restricted epitopes (246).
Whereas, preserving HLA-C and/ or HLA-E (which interact with NK cell inhibitory receptors) in
the surface of HIV-1 infected cells, prevent efficient killing by NK cells (247). Furthermore, it
has been shown that HIV-1 Nef impairs NK cell activity by down-regulating the expression of
ligands (MICA, ULBP1, and ULBP2) for the NKG2D NK cell activation receptor (248).
During early stages of HIV-1 infection, several extracellular components of the innate
immune system, inclusive of pro-inflammatory cytokines and chemokines, have been shown to
increase in the plasma of HIV infected individuals. For example, the plasma levels of type I IFN,
TNFα, IL-6, MCP-1, IFNγ, and IL-10 have all been found to be upregulated at various stages
following HIV-1 infection (249). These innate immune responses however have not been
associated with control of viremia, indicating that the rapid activation of systemic cytokine
cascade is not a prerequisite for viral clearance. Rather, it has been suggested that the induction
of these pro-inflammatory mediators may promote HIV-1 replication and suppress the antiviral
effect of type I IFNs (249). In addition, the CC chemokines MIP-1α (CCL3), MIP-1β (CCL4),
and RANTES (CCL5) produced from activated macrophages, DCs, T-, NK-, and γδ T-cells have
all been shown to play a role during HIV-1 infection. By binding and down-modulation of the
CCR5 HIV-1 co-receptor, these CC chemokines have been shown to prevent HIV-1 infection in
vitro and SIV infection in vivo (235). It has been shown that induction of these 3 CC chemokines
following vaccination of rhesus macaques with SIVgp120 and p27, inversely correlated with
proportion of cells expressing CCR5 co-receptor (250), suggesting that vaccination strategies
that up-regulate these CC chemokines may prevent HIV-1 infection.
43
Several intracellular molecules have also been found to play critical roles during HIV-1
infection. For example, the type I IFN induced nucleic acid editing enzyme, APOBEC3G, has
been shown to prevent HIV-1 viral replication (251). APOBEC3G is a cytosine deaminase that
converts cytidine to uridine in single stranded proviral DNA, which have been found to induce
hyper-mutation of the HIV-1 genome, rendering them non-functional and inhibiting viral
replication (252). This innate mechanism of resistance to HIV-1 infection has been found to be
counteracted by HIV-1 viral infectivity factor (vif) which prevents incorporation of APOBEC3G
into the virion and rapidly induces APOBEC3G ubiquitination and proteasomal degradation
(253). Induction of APOBEC3G in immune cells may represent a novel strategy for the
+
development of next generation HIV-1 vaccines. APOBEC3G upregulation in DCs and CD4 T
cells has been found to be mediated by several mechanism, including ligation of CCR5 (via MIP1α) and CD40 (via CD40L), and by the 70-kDa heat shock protein (HSP70) (254). Recently,
APOBEC3G has been shown to be induced in CD4+ memory T cells of rhesus macaques
following immunization with SIVgp120 and CCR5 peptides linked to HSP70 (255).
Furthermore, mucosal challenge with SIVmac251 showed a significant increase in APOBEC3G
+
+
mRNA in the CD4 CCR5 circulating memory T cells and the draining iliac lymph node cells in
the immunized uninfected macaques, suggesting a protective effect exerted by APOBEC3G
(255).
In addition to APOBEC3G, the family of tripartite motif (TRIM) proteins is another
example if type I IFN induced genes that have also been shown to restrict HIV-1 replication. In
rhesus macaques, TRIM-1α has been shown to block the early replication steps of HIV-1 and
other retroviruses by targeting the viral capsid (CA) protein for degradation (256). Moreover, it
has also been shown that the type I IFN induced protein, Tetherin (BST-2, also called CD317),
44
can also restrict HIV-1 by preventing the release of newly synthesized virions from HIV-1
infected cells, a mechanism that is counteracted by HIV-1 vpu protein, which targets the
transmembrane domain of tetherin for ubiquitin dependent proteasomal or lysosomal degradation
(257). HIV-1 virus has also developed several mechanisms that prevent type I IFN activation in
general. For example, HIV-1 vpr and vif proteins have been shown to target IRF-3 for
degradation in T cells, a mechanism that results in inhibition of the antiviral response and IFNβ
synthesis (258). Furthermore, the HIV-1 encoded protease was also found to play a role in the
inhibition of type I IFN by enhancing IRF-3 phosphorylation and thus preventing the induction
of ISG (259). Thus, despite these potent innate effector mechanisms, HIV-1 developed several
mechanisms to evade these innate responses.
These facts indicate that the innate immune response to HIV-1 is complex and represent a
novel mechanism in preventing HIV-1 transmission or controlling virus replication until the
development of an effective adaptive immune response. It also indicates that, harnessing the
innate immunity could be a valid strategy for the development of future HIV-1 vaccines.
2.1.3.2. Adaptive immune responses to HIV-1:
HIV-1 is predominantly transmitted through mucosal tissues, targeting and destroying
+
+
50% of CD4 CCR5 T cells within two weeks following the infection, setting the base for
progressive immunodeficiency (260). As mentioned above, the immune system recognizes and
initially controls HIV-1 replication but is incapable of fully eradicating the virus (232). HIV-1+
specific CD8 cytotoxic T cell responses have been detected at early stages following control of
primary viremia (261), implicating an important role for CTL responses in immune control of
viral replication. Several reports have also shown that cellular immune responses to HIV-1 are a
45
driving force for HIV-1 viral evolution (232), indicating a selective pressure by these cellular
+
immune responses. The Gag-specific CD8 T cell responses have been shown to inversely
correlate with HIV-1 RNA levels in chronically infected individuals (262). In addition, genetic
association studies have demonstrated that specific human leukocyte antigen (HLA) alleles are
closely associated with HIV-1 RNA levels (263). Moreover, it has been shown that HLAmediated escape mutations correlate with sites of inter- and intra-HIV-1 subtype variability
+
(264). Furthermore, depletion of CD8 T cells abrogated the control of SIV replication during
both acute and chronic infection (265).
The important role of cellular immune responses to HIV-1 has been further confirmed by
several pre-clinical and clinical vaccine studies (208). For example, several reports on SIV
infected rhesus monkeys have shown that vaccine strategies that elicit potent cellular immune
responses can reduce the setpoint viral load in specific SIV challenge models (266, 267). It is
+
worth mentioning here that the CD8 T cell, both natural or vaccine-elicited, responses are not
sufficient to completely prevent or eliminate the viral infection, however, by potentially lowering
the viral load set points, these cellular immune responses may help in minimizing both the rate of
disease progression in infected individuals and the transmission of the infection to other
individuals (268).
The role of humoral immune responses to HIV-1 has also been studied extensively, and is
again in the forefront based primarily upon results using HIV vaccines using the env gene to
generate neutralizing antibody responses that can be protective to some vaccinees (269).
Antibody responses of different isotypes to HIV-1 proteins env, gag, and pol have been found to
be elicited early following HIV-1 infection. Several reports have indicated that the initially
46
induced antibody response to HIV-1 is ineffective in controlling virus replication during acute
HIV-1 infection (270). HIV-1 specific IgM and IgG antibody responses to the envelope
glycoprotein (Env) (anti-pg41 (appeared at 13 day) and anti-gp120 (appeared at 28 day)) have
been detected in HIV-1 infected individuals; however, these responses are non-neutralizing and
have been shown to have a limited impact on acute-phase viremia (270). In addition, IgM and
IgG antibody responses to gag (appear at 18 day) and integrase (appear at 53 day) have also
been detected, however these responses have been found to have limited impact on viral RNA
level (231). Broadly reactive neutralizing antibody responses to HIV-1 Env have been suggested
to be the most important correlate of protection against HIV-1 infection. Recent studies by
Barouch et al group revealed that virus vaccine-elicited neutralizing antibody responses to Env
protect against acquisition of fully heterologous, neutralization-resistant SIV challenges in rhesus
monkeys (267), suggesting that neutralizing antibody responses may contribute significantly to
HIV-1 control and prevention. However, in chronic infection, neutralizing antibody responses to
Env are rarely generated and have been shown to appear several months (12 weeks) after the
HIV-1 infection (270). In addition, these HIV-1-specific anti-envelope responses were primarily
directed to gp120 and found to be predominantly IgG1 subtype, indicating that at chronic HIV-1
infection, the T helper responses were skewed to Th2 (271). Further, IgG2, IgG3, and IgG4
antibodies against gp120 were also detected, but are generally less often and not associated with
control of viremia. Furthermore, the range of epitopes targeted by these initial neutralizing
antibodies is very narrow allowing rapid viral escape (272). Several studies have shown that Env
neutralizing antibodies are directed against the monomeric gp120, rather than targeting the intact
HIV-1 Env trimer on the virion surface (273). In addition, the conserved HIV-1 CD4-binding
site, the chemokine receptor binding site, and key epitopes in the membrane proximal external
47
region (MPER) of gp41, are all targets for broadly reactive neutralizing antibodies; however,
these sites were found to be hidden or mutated and only exposed during receptor binding (232,
272). Furthermore, mucosal HIV-1-specific neutralizing IgA antibodies of unknown specificity
have been detected in genital secretion of high-risk HIV-1 uninfected sex workers and found to
correlate with subsequent protection from HIV-1 acquisition (274). Thus, development of
vaccine platforms that induce broadly reactive neutralizing antibodies that target the CD4binding site, glycan on the surface of pg120, and the MPER region of the gp41, is a primary area
of research for future antibody-based HIV-1 vaccines.
Several studies in rhesus monkey have shown that administration of high doses of
broadly reactive monoclonal antibodies can block transmission of SIV (275). More recently, the
results from the RV144 phase III “Thai” trial (four priming injections of a recombinant
canarypox virus expressing gp120, gag and protease as priming vaccination (ALVAC-HIV)
followed by a two booster injections of a recombinant glycoprotein 120 subunit vaccine
(AIDSVAX B/E)) suggested that a prophylactic HIV-1 vaccine is possible and protection from
HIV-1 infection can be achieved with vaccine strategies that can induce more potent T and B
cells responses to HIV-1-derived antigens, with gp120 being primarily implicated in this study
(269). Moreover, the results from this trial suggest that the vaccine induces a weakly protective
effect against viral acquisition but not viral control after infection (276), suggesting a role for the
elicited binding antibodies to gp120 protein.
2.1.4. HIV-1 vaccine trials
The best hope of controlling HIV-1 epidemic is through the development of a successful
prophylactic vaccine. Previously performed HIV-1 vaccine studies have relied primarily in the
48
induction of either neutralizing antibody or T cell-mediated immune responses, however, most of
these responses have largely failed to protect from HIV-1 infections. More recently, it has been
shown that vaccine regimens that can induce both cellular and humoral immune responses may
prevent from HIV-1 infection in human (269).
Since Edward Jenner’s discovery of vaccination to prevent smallpox, elicitation of
pathogen-specific antibody responses was regarded as the best correlate of protection. Utilizing
this concept, two HIV-1 vaccine efficacy studies were performed to date. The first study utilized
purified monomeric envelope glycoprotein (Env gp120) subunit vaccine in order to generate
HIV-1-specific antibody responses. The result from this trial revealed that antibody responses
induced by this vaccine were unable to neutralize primary virus isolates and to protect from HIV1 infection (277). Another two phase III studies known as AIDSVAX (VAX003 and VAX004)
(conducted by VaxGen in 2003) were performed to evaluate the protective efficacy of alum
adjuvanted Env gp120 protein. The results of these two studies have also indicated that rgp120
vaccination does not provide protection against HIV-1 infection in human (clinical isolates were
highly resistant to neutralization and the vast majority of antibodies induced by vaccination with
monomeric Env proteins were directed to decoy epitopes with no neutralization abilities) (278),
confirming the need to develop more potent, broadly reactive, neutralizing antibody responses by
HIV-1 vaccines (279).
The induction of potent cell-mediated immune responses against HIV-1 has been the
focus in the past few years. Several reports have demonstrated that induction of potent CTL
responses is the primary correlate of protection against viral infection (280). In addition, the
result from early studies in NHP confirmed the importance of CTL responses in controlling HIV1 infection (265). Moreover, CTL responses (predominantly gag-specific responses) in a group
49
of HIV-1 infected patients who control viral replication without therapy, called “elite
controllers”, indicated a direct correlation between strong CTL responses and control of viremia
(281), justifying the development of vaccination approaches that elicit potent CTL responses.
Studies in NHPs challenged with a hybrid SHIV virus utilizing different recombinant
adenovirus platforms supported a rAd5-based HIV-1 vaccine clinical trial (known as STEP®
trail; HVTN 502) performed by Merck Research Laboratories. Recombinant Ad5 vaccines
expressing several HIV-1 antigens (HIV-1 clade B Gag, Pol and Nef) clade B Gag were utilized
in a homologous prime-boost regimen at months 0, 1, and 6. The results from the trial revealed
that cellular immune responses specific for the HIV-1 antigens were induced in the majority
(73%) of vaccinated individuals (209, 217). The inductions of these cellular immune responses
were completely blunted in individuals with pre-existing Ad5-specific immunity. The STEP
study was terminated in 2007 due to lack of efficacy, as no evidence of vaccine induced
protection (preventing acquisition of infection or facilitating control of HIV-1 replication postinfection) was observed in vaccinees (210). Similar clinical trial conducted in Thailand, known
as Phambili (HVTN 503), was also terminated because it utilized the same rAd5 vaccine vectors
in a population with higher levels of pre-existing Ad5-specific immunity. While it was initially
thought that pre-existing immunity to Ad might enhance HIV infection rates in the STEP
vaccinees, subsequent studies have noted that circumcision rates and/or other confounding
factors likely contributed to the skewed infection rates noted between placebo and Ad
vaccinated, Ad immune participants in the trial (282-284).
The negative results from antibody-based and the Merck rAd5-based trials led to
development of different strategies aiming to develop more potent humoral and cellular immune
responses to HIV-1 antigens. Heterologous prime-boost vaccination strategy has been shown to
50
provide higher magnitude (and quality) B- and T-cell responses, compared to homologous
prime-boost vaccination approaches. More recently, it has been shown that, a heterologous
prime-boost vaccination against HIV-1 might be an effective strategy to prevent HIV-1 infection
in humans. The results from the clinical trial, RV144, showed 31% reduction in infection
acquisition, but not control of viremia, in vaccinees compared to controls (269). The ALVAC
prime-gp120 boost vaccine regimen induced strong transient Env-specific antibody responses
+
+
and Env-specific CD4 T cell responses, but no significant HIV-specific CD8 T cells
responses. This strongly suggests a critical role for humoral immune responses in preventing
+
viral acquisition and potentially the need to elicit more potent CD8 T cells responses to control
viral replication post infection in more vaccinees. Importantly, similar protection levels were also
observed in NHP studies.
Recent heterologous prime-boost (rAd26 prime/ modified vaccinia Ankara (MVA) boost)
studies in NHPs have shown a critical role for the elicited neutralizing antibody responses to Env
to protect against acquisition of fully heterologous, neutralization-resistant SIV challenges in
rhesus monkeys (267). In another study, a prime-boost (A plasmid DNA prime/ rAd5 boost)
vaccine regimen was evaluated for ability to protect monkeys from infection by SIVmac251 or
SIVsmE660 isolates after repeat intrarectal challenges. The prime-boost vaccination protected
50% of vaccinated monkeys from acquisition of SIVsmE660, but had a minimal effect on viral
loads of successfully infected monkeys. Notably, low levels of neutralizing antibodies and an
+
envelope-specific CD4 T cell response were associated with vaccine protection in these
monkeys (285). These studies begin to elucidate the mechanisms of vaccine protection against
51
HIV-1 and highlight the need to develop vaccine strategies capable of eliciting more potent
humoral and CTL immune responses in future trials of HIV-1 vaccines.
The previous data described above laid the foundation for the original body of work
described herein. While it has become increasingly accepted that use of the rAd5 vaccine vectors
elicits potent antibody and cellular immune responses to specific antigens as compared to
immunizations using other vaccine platforms, the results from STEP trial suggest that a more
potent vaccine capable of inducing greater levels of antigen specific adaptive immune responses
may demonstrate greater efficacy to prevent HIV-1 infection. The work in this chapter attempts
to describe the development of a novel rAd5-based HIV-1 vaccine approach that targets the
SLAM family of receptors signaling in innate immune cells by expressing an adaptor molecule,
known as EAT-2, identified to be critical for regulating signal transduction and proinflammatory cytokines responses downstream of SLAM receptors in innate immune cells.
The SLAM family of receptors currently comprise six distinct members, respectively
named SLAM (CD150), 2B4 (CD244), Ly9, CD84, NTB-A (natural killer, T and B cell antigen;
Ly108 in the mouse) and CRACC (CD2-like receptor activating cytotoxic cells) (286). These
receptors are expressed mainly in cells of the hematopoietic lineages, inclusive of innate and
adaptive immune cells (149, 287). All SLAM members except 2B4, (which interacts with CD48)
are self ligands, and can be triggered by homophilic interactions through their respective
extracellular domains to initiate intra-cellular signaling via recruitment of specific adaptors (287290). The SLAM-associated protein (SAP) family of adaptors includes three members named
SAP, EAT-2, and EAT-2-related transducer (ERT; ERT is however a non-functional pseudogene in humans). These adaptors associate with phosphorylated tyrosine-based motifs
(‘immunoreceptor tyrosine based switch motifs’ (ITSM)) in the cytoplasmic domains of SLAM
52
receptors with high affinity and specificity. Once bound, these adaptors either augment or inhibit
SLAM induced intracellular signaling in a variety of immune cells (286). EAT-2 and ERT
transduce SLAM initiated signals via phosphorylation of tyrosine residues directly located in
their short carboxyl-terminal tails (165). In contrast, the SAP adaptor regulates SLAM signaling
by recruiting the protein tyrosine kinase FynT.
Various reports indicate a possible role for the binding of SLAM (CD150) receptor in the
immunological synapse, whereby SLAM receptor activation acts as a costimulatory molecule
facilitating the activation of DCs and macrophages. For example, in CD40L-activated human
DCs, antibody-mediated ligation of SLAM receptor augmented the secretion of proinflammatory cytokines such as IL-12 and IL-8, but not IL-10 (291). Furthermore, the SLAM
receptor was also found to play a role in the production of IL-6 and IL-12 by mouse peritoneal
macrophages (292). In addition, macrophages derived from SLAM-deficient mice show a
marked reduction in secretion of IL-12, TNF, and nitric oxide (292). Since EAT-2 is the only
known SLAM-associated adaptor protein expressed in DCs and macrophages, it has been
proposed that EAT-2 facilitates SLAM dependent proinflammatory cytokine expression in these
cell types (290).
Relative to vaccine augmentation strategies, we note that the TLR adaptor MyD88 also
facilitates cytokine gene expression during TLR dependent signaling, and that over-expression of
MyD88 from DNA based vaccines facilitated the induction of antigen specific adaptive immune
responses by the vaccine platform (293, 294). Based upon these facts, we hypothesized that
expression of EAT-2 from a vaccine platform could also augment innate immune responses,
potentially resulting in improved APC functions in vivo and consequently improvement in the
induction of adaptive immune response generated against a co-expressed target antigen. In this
53
study, we confirmed that an adenovirus based vaccine expressing the SLAM adaptor molecule
EAT-2 facilitates the induction of several arms of the innate immune system, and that these
inductions positively correlate with an improved ability of the vaccine to induce stronger cellular
immune responses to a co-expressed antigen. Our results highlight for the first time the use of a
SLAM immune regulatory pathway component to improve vaccination efficacy.
54
2.2.
Results
EAT-2-expressing Ad vectors enhance Ad vector induced innate immune responses in vivo.
We constructed an E1 and E3 deleted ([E1-])Ad vector specifically designed to express the
murine homologue of the SLAM adaptor protein EAT-2. These EAT-2 expressing Ad vectors
were fully viable, grew to high titers, and were purified and quantified exactly as done for
conventional [E1-]Ad vaccines (184). To analyze the maximal impact that Ad-EAT2 expression
might have upon innate immune responses, we administered Ad5-EAT2 or Ad-Null control into
C57BL/6 mice and measured the levels of cytokines and chemokines by utilizing 23-plex
multiplex based assay in the plasma at 3, 6, 10, 24, and 48 hours post injection (hpi). We targeted
these time points to evaluate the kinetics of EAT-2 effects on the induction of pro-inflammatory
cytokines and chemokines occurs after Ad5-EAT2 vector administration. Administration of the
Ad5-EAT2 vector into C57BL/6 mice resulted in induction of significantly higher plasma levels
of, IL-1a, IL-5, IL-12p70, IFNγ, G-CSF, and GM-CSF as directly compared to mice identically
treated with an Ad-Null control vector (Figure 1a). Interestingly, the levels of EOTAXIN, IL-10,
and KC were significantly reduced in Ad5-EAT2 injected mice compared to Ad-Null injected
controls (Figure 1b). The levels of IL-1β, IL-12p40, IL-6, IL-9, MCP-1, MIP-1α, MIP-1β, and
RANTES were also significantly induced by Ad5-EAT2 vectors; however, these levels were not
statistically different between Ad5-EAT2 and Ad-Null injected mice (data not shown). We also
measured the levels of IL-3 and IL-4, however, no significant inductions were observed in mice
injected with both Ad-EAT-2 and Ad-Null vectors (data not shown).
55
Figure 1: Systemic administration of EAT-2 expressing adenovirus vector induces cytokine
and chemokines responses. C57BL/6 mice (n=4) were either mock injected, or intravenously
injected with 7.5×10
10
vps of either Ad-Null or Ad-EAT2 vectors. Plasma was harvested at 3,
6, 10, 24, and 48 hours after virus injection. Cytokine induction was evaluated using a
multiplexed bead array based quantitative system. The bars represent mean ± SD. Statistical
analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test,
p<0.05 was deemed a statistically significant difference. ** denotes p<0. 01, *** denotes p<0.
001- Statistically different from mock injected animals).
56
Figure 1: Systemic administration of EAT-2 expressing adenovirus vector induces cytokine
and chemokines responses.
57
To first determine what innate immune cell types may be transduced by the Ad vectors
expressing EAT2, (and potentially be responsible for the enhanced cytokine responses noted in
Fig 1), we analyzed Ad vector transduction of several important classes of innate immune cells
found in the spleen by flow cytometry. Utilizing an Ad vector expressing a tracking antigen
(GFP) we confirmed that Ad vectors can transduce dendritic cells, NK cells, as well low levels of
T cells upon administration into mice (Figure 2a-d). Interestingly, we found that the highest
+
+
subset of DCs that were transduced by Ad-GFP vectors were CD11c , CD8α , a distinct subset
of dendritic cells shown to play a critical role in shaping adaptive immune responses after
vaccination (190) (Figure 2a). Additionally, we confirmed that after identical administrations of
the Ad-EAT2 vector, EAT-2 gene expression was occurring in spleen cells as well (Figure 2e).
Figure 2: Transduction efficiency of innate immune cells by Adenovirus vectors expressing
transgenes. C57BL/6 mice (n=3) were either mock injected, or intravenously injected with 7.5 ×
10
10
vps of Ad-GFP. Splenocytes were harvested 6 hours post-injection and GFP expressing
cells were detected and identified by gating on FITC+ cells using a LSR-II flow cytometer. (A)
+
+
+
Ad-GFP Transduced CD11c CD8α dendritic cells. (B) Ad-GFP transduced CD11c CD8α
dendritic cells. (C) Ad-GFP transduced CD3- NK1.1+ cells. (D) Ad-GFP transduced CD3
+
-
+
CD8 T cells. (E) Quantitative RT-PCR for EAT-2 transcript derived from the spleen of Mock,
Ad-GFP, or Ad-EAT2 injected C57BL/6 mice. The bars represent mean ± SD. Statistical
analysis was completed using student t-test, p<0.05 was deemed a statistically significant
difference. * denotes p<0. 05- Statistically different from mock injected animals.
58
Figure 2: Transduction efficiency of innate immune cells by Adenovirus vectors expressing
transgenes.
59
It is widely appreciated that Ads enhance innate immune cell effector functions (295,
296). To additionally evaluate the phenotype of immune cells following exposure or transduction
by the Ad vector expressing EAT-2, we analyzed the expression of the lymphocyte activation
marker CD69 as well as IFN-γ production in various immune cells shortly after administration of
Ad-EAT2 into C57BL/6 mice. Our results confirmed that Ads, in general, significantly induce a
rapid activation of NK and NKT cells in both PBMCs and spleens, as confirmed by the presence
of increased percentages of CD69 expressing NK and NKT cells in Ad-GFP treated mice (Figure
3a and b). Specifically, injection of Ad-EAT2 induced significantly higher numbers of CD69
expressing NK and NKT cells, both in PBMCs and splenocytes at 6 hpi, as compared to the AdGFP control vector (Figure 3a and b).
Figure 3: Ad-EAT2-mediated activation of innate and adaptive immune cells in vivo.
C57BL/6 mice (n=4) were either mock injected, or intravenously injected with 7.5 × 10
10
vps of
either Ad-GFP or Ad-EAT2. CD69 expression by PBMCs (A) and splenocytes (B) derived NK,
+
+
+
-
NKT, CD3 CD8 , CD3 CD8 , and B cells was evaluated 6 hours after virus injection. PBMCs
and Splenocytes were harvested, stained and sorted on a LSRII flow cytometer. The bars
represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a
student- Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference.
* denotes p<0.05, ** denotes p<0. 01, *** denotes p<0. 001, # denotes p<0. 001- Statistically
different from mock injected animals.
60
Figure 3: Ad-EAT2-mediated activation of innate and adaptive immune cells in vivo.
61
At 48 hpi, CD69 expression on NK cells remained significantly higher (p<0. 05) only in
PBMCs derived from Ad-EAT2 injected mice as compared to Ad-GFP injected mice (Figure 4a).
All other differences previously noted at 6 hpi had dissipated by 48 hpi (Figure 4c), suggesting
that expression of EAT-2 by Ad vaccines transiently improves the induction of these responses.
Figure 4: Ad-EAT2-mediated activation of innate and adaptive immune cells in vivo.
C57BL/6 mice (n=4) were either mock injected, or intravenously injected with 7.5 × 10
10
vps of
either Ad-GFP or Ad-EAT2. CD69 expression by PBMCs (a and b) and splenocyte (c and d)
+
+
+
-
derived NK, NKT, CD3 CD8 T cells, CD3 CD8 T cells, and B cells was evaluated 48h after
virus injection. PBMCs and Splenocytes were harvested, stained and sorted on a LSRII flow
cytometer. The bars represent mean ± SD. Statistical analysis was completed using One Way
ANOVA with a student- Newman-Keuls post-hoc test, p<0.05 was deemed a statistically
significant difference. * denotes p<0.05, ** denotes p<0. 01, *** denotes p<0. 001- Statistically
different from mock injected animals.
62
Figure 4: Ad-EAT2-mediated activation of innate and adaptive immune cells in vivo.
63
Treatment with either Ad-EAT2 or Ad-GFP induced significantly elevated numbers of
IFNγ+ NK cells at both 6 and 48 hpi (p<0. 001 and p<0. 05, respectively). However, no
significant differences were observed between the control and experimental viruses, suggesting
that overexpression of EAT2 cannot induce additional inductions of IFNγ relative to the ability
of the Ad vector itself (Figure 5).
Figure 5: IFNγ production from NK cells 6 and 48 hours after Ads injection. C57BL/6 mice
(n=4) were either mock injected, or intravenously injected with 7.5× 10
10
vps of either Ad-GFP
or Ad-EAT2 for 6 hpi (a) or 48 hpi (b). Splenocytes were harvested and incubated at 37°C for 5
hours in the presence of Golgi plug. IFNγ intracellular staining was performed and cells were
sorted on a LSRII flow cytometer. The bars represent mean ± SD. Statistical analysis was
completed using One Way ANOVA with a student- Newman-Keuls post-hoc test, p<0.05 was
deemed a statistically significant difference. *denotes p<0.05, *** denotes p<0. 001- Statistically
different from mock injected animals.
64
Figure 5: IFNγ production from NK cells 6 and 48 hours after Ads injection.
65
Since the SLAM family of receptors are expressed in various innate and adaptive
immune cells (149) and the activation of T cell and/or B cells can be initiated or accentuated by
innate immune system activation , we sought to analyze adaptive immune cell responses shortly
after administration of Ad-EAT2. Our result confirmed that Ad vector administration in and of
+
+
+
-
itself induces a rapid activation of CD3 CD8 T-cells CD3 CD8 T-cells, and B cells, Ad
dependent responses that can all be further accentuated with expression of EAT-2. For example,
injection of Ad-EAT2 resulted in significantly higher numbers of splenic CD69 expressing
+
+
+
-
CD3 CD8 T cells (p<0. 01), CD3 CD8 T cells (p<0. 01), and B cells (p<0. 001) at 6 hpi
(Figure 6b) as compared to the numbers of these cells being induced by the control Ad vector.
Figure 6: Ad-EAT2-mediated activation of innate and adaptive immune cells in vivo.
C57BL/6 mice (n=4) were either mock injected, or intravenously injected with 7.5 × 10
10
vps of
either Ad-GFP or Ad-EAT2. CD69 expression by PBMCs (a) and splenocytes (b) derived NK,
+
+
+
-
NKT, CD3 CD8 , CD3 CD8 , and B cells was evaluated 6 hours after virus injection. PBMCs
and Splenocytes were harvested, stained and sorted on a LSRII flow cytometer. The bars
represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a
student- Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference.
* denotes p<0. 05, ** denotes p<0. 01, *** denotes p<0. 001, # denotes p<0. 001- Statistically
different from mock injected animals.
66
Figure 6: Ad-EAT2-mediated activation of innate and adaptive immune cells in vivo.
67
+
+
+
-
At 48 hpi, the percentage of splenic CD69 expressing CD3 CD8 , CD3 CD8 T cells,
and B cells derived from Ad-EAT2 and Ad-GFP injected mice remained significantly higher
(p<0. 001) over mock injected mice; however, no statistical differences were observed between
the control and experimental Ad viruses at this time point (Figure 4d). We also evaluated the
percentage of CD69 expressing cells in PBMCs from the same animals. At 6 hpi, we observed a
significantly (p<0. 05) higher percentage of CD69 expressing total lymphocytes [as defined
previously] in Ad-EAT2 injected mice as compared to Ad-GFP injected mice. When analyzing
specific lymphocyte subsets, we observed a significant increase in the percentage of CD69
+
-
expressing CD3 CD8 T cells isolated from Ad-EAT2 injected mice as compared to the
percentage of identical cells isolated from Ad-GFP injected mice (p<0. 05) (Fig 6a). Higher
+
-
activation of CD3 CD8 T cells and B cells were observed in PBMCs derived from Ad-EAT2
injected mice as compared to the mock infected mice, however no statistically significant
differences in activation levels were observed between the control and experimental Ads in these
cell types (Figure 6a). By 48 hpi in PBMCs any statistically significant differences between
control and experimental Ad vector injected animals had resolved (Figure 4b). Simultaneous
administration of antigens with adjuvants can stimulate the innate immune system to
significantly improve the adaptive immune responses to the antigenic target (143, 297). To
investigate whether the enhanced innate immune responses promoted by Ad mediated expression
of EAT-2 could influence the adaptive immune responses to a co-administered antigen, we
immunized Balb/c or C57BL/6 mice with both an Ad-based vector expressing the HIV-1 clade B
Gag protein (HXB2) along with the Ad-EAT2 vector, and a control vector (Ad-GFP). We
performed initial dose curve studies to identify the lowest dose of Ad-HIV/Gag that generated
68
detectable Gag-specific cellular immune responses in the two distinct strains of mice. As a result,
6
8
we identified an Ad-HIV/Gag dose of 5 x10 vps/mouse for Balb/c mice, and 5 x10 vps for
C57BL/6 mice as the most relevant experimental doses for these studies (data not shown). Six
6
days after Balb/c mice were intramuscularly injected with 5x10 of Ad-HIV/Gag mixed with
equivalent amounts of either the control Ad-GFP vector, or Ad-EAT2, we were able to detect
+
heightened Gag specific, tetramer-positive CD8 T cells in PBMCs derived from mice coimmunized with Ad-HIV/Gag + Ad-EAT2 as compared to Ad-HIV/Gag+ Ad-GFP coimmunized mice (Figure 7a ). At 14 dpi , PBMCs (p<0. 05) and splenocytes (p<0. 05) derived
from mice co-immunized with Ad-HIV/Gag + Ad-EAT2 contained higher numbers of Gagspecific tetramer-positive CD8+ T cells as compared to the respective cell populations isolated
from control mice (Figure 7b and c).
Figure 7: HIV-Gag specific cellular immune responses elicited by Ad-HIV/Gag and AdEAT2 co-immunization. Balb/c mice were co-immunized intramuscularly in the tibialis anterior
with equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad-EAT2 (total of
7
1×10 vps). At 6 dpi, peripheral blood mononuclear cells (PBMCs) (A) were collected from the
immunized mice and stained with a PE-conjugated AMQMLKETI tetramer complex (A). At 14
dpi, mice were sacrificed and PBMCs (B) or splenocytes (C) were harvested and stained with a
PE-conjugated H2-Kd-AMQMLKETI tetramer complex together with an APC-conjugated antiCD3 and FITC-conjugated anti-CD8 antibodies. The bars represent mean ± SD for six mice per
group (pool of two for PBMCs) for virus injected and three mice for naïve animals. Statistical
analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test,
69
p<0.05 was deemed a statistically significant difference. * denotes p<0. 05, ** denotes p<0. 01Statistically different from mock injected animals.
Figure 7: HIV-Gag specific cellular immune responses elicited by Ad-HIV/Gag and AdEAT2 co-immunization.
70
Following ex vivo stimulation with the immunodominant Gag peptides AMQMLKETI
(QBI# 304754, for Balb/c mice) or QBI# 304796 (for C57BL/6 mice), splenocytes derived from
mice co-immunized with Ad-HIV/Gag and Ad-EAT2 contained significantly (p<0. 001)
increased numbers of Gag-specific, IFN-γ secreting cells (Figure 8a and b, respectively). We also
observed significantly increased numbers of IL-2 secreting, HIV-Gag peptide specific (QBI#
304754) splenocytes derived from Balb/c mice co-immunized with Ad-HIV/Gag + Ad-EAT2 as
compared to Ad-HIV/Gag+ Ad-GFP co-immunized mice (p<0. 01) (Figure 8a).
Figure 8: HIV-Gag specific cellular immune responses elicited by Ad-HIV/Gag and AdEAT2 co-immunization. Balb/c mice (n=6) (a) or C57Bl6 mice (n=4) (b) were co-immunized
intramuscularly in the tibialis anterior with equivalent viral particles of Ad-HIV/Gag mixed with
7
9
either Ad-GFP or Ad-EAT2 (total of 1×10 vps for Balb/c mice and total of 1×10 vps for
C57Bl/6 mice mixed prior to injection). At 14 d. p. i., splenocytes were harvested and stimulated
ex vivo with the immunodominant peptides (AMQMLKETI) for Balb/c and QBI# 304796
(EAMSQVTNSATIMMQ) for C57Bl/6. Spot forming cells (SFCs) were quantified using and
ELISPOT reader. Data are presented as mean ± SD. Statistical analysis was completed using
Two-Way ANOVA with a Bonferroni post-hoc test, p<0.05 was deemed a statistically
significant difference. ** denotes p<0. 01, *** denotes p<0. 001. Representative data from two
independent experiments are shown.
71
Figure 8: HIV-Gag specific cellular immune responses elicited by Ad-HIV/Gag and
Ad-EAT2 co-immunization.
72
EAT-2 expression during HIV-Gag vaccination also facilitated a broadened induction of
HIV-Gag specific T cell clones, as we observed increased numbers of Gag-specific IFN-γ and
IL-2 co-secreting splenocytes responding to an expanded variety of HIV-Gag specific peptides
present within the full HIV-Gag protein (i.e.: QBI# 304742, 304769, 304779, 304800, and a
peptide pool (304790, 403808, and 304826) (Figure 9a and b).
Figure 9: Analysis of the breadth of Gag-responses. Balb/c mice were co-immunized
intramuscularly with equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad7
EAT2 (total dose of 1×10 vps mixed prior to injection). At 14 d. p. i. , animals were terminally
sacrificed, and splenocytes were harvested and stimulated ex vivo with 15mer HIV-Gag derived
peptides QBI#304724, (SLYNTVATLYCVHQR), QBI#304753(GHQAAMQMLKETINE),
QBI#304754 (AMQMLKETINEEAAE), QBI#304769 (PVGEIYKRWIILGLN), QBI#304779
(VDRFYKTLRAEQASQ), QBI# 304800 (GNFRNQRKTVKCFNC), or pool of three peptides
(BQI# 304790, 304808, and 304826), and IFNγ (a) and IL-2 (b) ELISPOT assays were
completed. Bars represent mean ± SD. Statistical analysis was completed using Two-Way
ANOVA with a Bonferroni post-hoc test, p<0.05 was deemed a statistically significant
difference. * denotes p<0. 05, ** denotes p<0. 01, *** denotes p<0. 001, # denotes p<0. 001Statistically different from naive animals.
73
Figure 9: Analysis of the breadth of Gag-responses.
74
In a more global analysis as to the extent of HIV-Gag peptide recognition promoted by
prior expression of EAT-2, we ex vivo stimulated splenocyte preparations derived from the
immunized mice with HIV-gag derived peptide pools, each pool containing 2-4 Gag specific
15mer peptides spanning the entire HIV-Gag protein sequence. We observed an increased
breadth in the immune recognition of HIV-Gag by co-expression of EAT-2, as the number of
HIV Gag-specific peptides that triggered cellular responses from splenocytes derived from the
Ad-HIV/Gag + Ad-EAT2 co-immunized mice were significantly increased, as compared to
similarly treated splenocytes derived from the Ad-HIV/Gag+ Ad-GFP control mice (Figure 10).
Figure 10: “(For interpretation of the references to color in this and all other figures, the
reader is referred to the electronic version of this dissertation)” Analysis of T cell epitope
responses of Balb/c and C57Bl/6 mice to HIV-Gag in Ad-HIV/Gag and Ad-EAT2 coinjected mice. Balb/c (n=6) (a) or C57BL/6 (n=4) (b) mice were co-immunized with equivalent
7
viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad-EAT2 (1×10 total vps for
9
Balb/c and 1×10 total vps for C57Bl/6 mice). At 14 dpi splenocytes were equivalently pooled
and IL-2 ELISPOT analysis was carried out by stimulating individual wells ex vivo with a pool
of 2-4 15mer peptides overlapped by 11, not including peptides included in Figure 4 and 5. SFCs
per million splenocytes are shown. The minimal threshold response is indicated by the line above
10.
75
Figure 10: “(For interpretation of the references to color in this and all other figures, the
reader is referred to the electronic version of this dissertation)” Analysis of T cell epitope
responses of Balb/c and C57Bl/6 mice to HIV-Gag in Ad-HIV/Gag and Ad-EAT2 coinjected mice.
76
CD8 T cell depletion studies suggested that the majority of the T cells responding to the
+
antigens were CD8 (Figure 11).
Figure 11: Cellular immune responses after CD8+ T cells depletion in Ad-HIV/Gag and Ad7
EAT2 co-immunized mice. At 14 dpi, splenocytes from vaccinated Balb/c mice (1×10 total
vps) were equivalently pooled (N=6 mice per treatment) and CD8+ cells were depleted using
5
magnetic beads. 5×10 splenocytes were added to each well and stimulated with the
immunodominant peptide AMQMLKETI. (a) A representative flow cytometric analysis before
+
+
and after CD8 T cell depletion is shown. Spots from CD8 un-depleted cells and CD8 depleted
cells were quantified using an automated ELISPOT reader (b and c). %SFC that are CD8- =
+
(#SFCs CD8 dep / #SFCs CD8 )*100 (d). The bars represent mean ± SD. Statistical analysis
was completed using student’s t-test.
77
Figure 11: Cellular immune responses after CD8+ T cells depletion in Ad-HIV/Gag and AdEAT2 co-immunized mice.
78
Published reports have shown that the increased presence of antigen specific T cells that
express several cytokines in response to antigens correlate with improved vaccine-induced
protective immunity, and positively correlate with the induction of long lived memory responses
(298, 299). To enumerate these "polyfunctional" T cells, we evaluated the expression of
+
cytokines in HIV-Gag-specific CD8 T cells generated after Ad-HIV/Gag and Ad-EAT2 co+
immunization. Six-color flow cytometry was used to enumerate the frequency of CD8 T cells
producing IFNγ, TNFα, and/or IL-2 after ex vivo stimulation with HIV-Gag specific peptides.
We observed statistically higher numbers of HIV-Gag-specific IFNγ-positive (p<0. 05) (Figure
+
12 b and c) and IFNγ/ TNFα-double positive (p<0. 05) (Figure 12 d and e) CD8 T cells derived
from Ad-HIV/Gag and Ad-EAT2 co-immunized mice as compared to mice vaccinated with the
+
control vaccines. When evaluating TNFα or IL-2 single positive CD8 T cells, we also observed
+
increased numbers of CD8 T cells that express TNFα or IL-2 derived from Ad-HIV/Gag and
Ad-EAT2 co-immunized mice as compared to Ad-HIV/Gag and Ad-GFP co-immunized mice;
however, with these numbers of samples these improved trends did not reach statistically
significant levels (data not shown).
Figure 12: Ad-HIV/Gag and Ad-EAT2 co-immunization increases the frequency of HIVGag specific CD8+ T cells. Balb/c (n=6) or C57BL/6 (n=4) mice were co-immunized with
7
equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad-EAT2 (1×10 total
9
vps for Balb/c and 1×10 total vps for C57Bl/6 mice). At 14 dpi, the mice were sacrificed and
lymphocytes were isolated from spleen. Multiparameter flow cytometry was used to determine
+
the total frequency of cytokine-producing CD8 T cells. (a) Representative example of the
79
+
gating strategy used to define the frequency of cytokine producing CD8 T cells. Gate were set
based on negative control (naïve) and placed consistently across samples. The total frequency of
+
CD8 T cells derived from Balb/c or C57Bl/6 mice expressing IFNγ (b and c, respectively) or
IFNγ and TNFα (a and e, respectively) is shown. The bars represent mean ± SD. Statistical
analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test,
p<0.05 was deemed a statistically significant difference. * denotes p<0. 05- Statistically
different from naive animals.
80
Figure 12: Ad-HIV/Gag and Ad-EAT2 co-immunization increases the frequency of HIVGag specific CD8+ T cells.
81
+
The direct measurement of in vivo functionality of CD8 T lymphocytes (CTL) to
specifically kill antigen presenting target cells provides a critical assessment as to their overall
+
functional capacity. To evaluate the in vivo cytolytic activity of the Gag-specific CD8 T
lymphocytes generated after Ad-HIV/Gag and Ad-EAT2 co-immunization, mice were coimmunized with Ad-HIV/Gag+ Ad-EAT2 or Ad-HIV/Gag+ Ad-GFP. 14 days after vaccination,
the two groups of mice were then injected with carboxyfluorescein succinimidyl ester (CFSE)labeled syngeneic splenocytes pulsed with the Gag derived peptides AMQMLKETI or QBI#
304796. The elimination of the peptide-pulsed splenocytes (CFSE
high
) was then examined by a
flow cytometry-based CTL assay (300). Our results demonstrated that HIV-Gag specific CTL
activities induced in mice co-immunized with Ad-HIV/Gag and Ad-EAT2 were significantly
higher as compared to control mice (p<0. 05 for Balb/c and p<0. 01 for C57Bl/6 mice) (Figure
13 a and b).
Figure 13: Increased cytolytic activity of the Gag-specific T-cell in vivo in Ad-HIV/Gag and
Ad-EAT2 co-immunized mice. Balb/c (n=6) (a) or C57BL/6 (n=4) (b) mice were coimmunized with equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad7
9
EAT2 (1×10 total vps for Balb/c and 1×10 total vps for C57Bl/6 mice). At 14 dpi, syngeneic
splenocytes were pulsed with either an irrelevant peptide (NYD-pep) and stained with 1µM
(CFSE
Low
) (or with the HIV-Gag specific peptides (AMQ peptide for Balb/c and QBI# 304796
for C57Bl/6 mice) and labeled with 10µM (CFSE
High
). Five hours after adoptive transfer into
either Naïve or immunized mice, splenocytes were harvested and sorted using a LSRII flow
cytometer. % CFSE positive cells were quantified using FlowJo software. % specific killing = 1-
82
((% CFSE
High
/ % CFSE
Low
) immunized / (% CFSE
High
/ % CFSE
Low
) non-immunized). **
denotes p<0. 01, *** denotes p<0. 001- Statistically different from naive animals. Representative
figure of two combined independent experiments is shown for Balb/c mice.
83
70
60
50
40
30
20
10
85
70
60
50
40
30
20
10
10
10
Figure 13: Increased cytolytic activity of the Gag-specific T-cell in vivo in Ad-HIV/Gag and
Ad-EAT2 co-immunized mice.
84
To investigate the mechanisms underlying the enhanced Gag-specific adaptive immune
responses generated after Ad-HIV/Gag and Ad-EAT2 co-immunization, we evaluated the
expression of markers associated with APC function, in this instance analyzing bone marrow
derived macrophages (BMDMs). Isolated BMDMs were initially infected with the Ad-EAT2 or
Ad-GFP vectors at escalating multiplicities of infection (5,000-50,000 vps/cell). Cells were then
analyzed for the surface expression of CD80 and CD86 co-stimulatory molecules by flow
cytometry. Mock infected BMDMs expressed low levels of CD80 and CD86 while infection
with the control Ad virus significantly induced the expression of CD80 and CD86 molecules
(Figure 14). Interestingly, Ad-EAT2 infection greatly enhanced the expression of CD80 and
CD86 as compared to BMDMs infected with the control Ad (Figure 14). These effects were not
seen if the BMDMs were infected with a UV irradiation-inactivated Ad-EAT2 vector,
confirming that EAT-2 gene expression was necessary for the effect (data not shown).
Figure 14: EAT2 overexpression augments CD80 and CD86 expression by bone marrow
derived macrophages. In vitro cultured murine BMDMs (500,000 cells/ well) were mock
infected or infected with the Ad-EAT2 or Ad-GFP control for 72 h at the multiplicity of infection
(MOI) shown. (A) Expression of GFP on BMDMs following infection with escalating doses of
Ad-GFP at 72 h. (B) Expression of CD80 on BMDMs 72 h postinfection with escalating doses of
Ad-EAT2 (black) or Ad-GFP (gray). (C) CD86 expression in BMDMs infected with various
doses of Ad-EAT2 or Ad-GFP at 72 h postinfection. Data are representative of three
independent experiments with similar results. Samples were plated in triplicate and are expressed
as mean ± SD (* P<0.05, ** P<0.01, *** P< 0.001 statistically different from uninfected cells).
85
Figure 14: EAT2 overexpression augments CD80 and CD86 expression by bone
marrow derived macrophages.
86
RAW264.7 cells were also infected with either the Ad-EAT2 or Ad control vector.
Similar to the results we obtained in BMDMs, Ad infection resulted in significant increases in
the expression of CD40, CD80, CD86, and MHC-II molecules (Figure 15). Importantly, AdEAT-2 infection resulted in significantly higher levels of CD40, CD80, CD86, and MHC-II
molecules as compared to cells infected with the Ad control virus (Figure 15).
Figure 15: Increased expression of CD40, CD80, CD86, and MHC-II in Ad-EAT2 infected
RAW264.7 cells. RAW264.7 cells (500,000 cells/ well) were mock infected or infected with
20,000 vector particles/ cell of Ad-EAT2 (black or Ad-Null (gray). Seventy-two hours later, cells
were stained with antibodies specific for CD40, CD80, CD86, and MHC class II, and analyzed
on an LSR-II flow cytometer. Data are representative of four independent experiments with
similar results. Samples were plated in triplicate and are expressed as mean ± SD (*** denotes
P< 0.001 statistically different from uninfected cells).
87
Figure 15: Increased expression of CD40, CD80, CD86, and MHC-II in Ad-EAT2
infected RAW264.7 cells.
88
2.3.
Discussion
The SLAM family of receptors modulate multiple innate and adaptive immune responses
through their intracellular signaling adaptors, SAP, EAT-2, and ERT (141, 290, 301). This report
provides evidence supporting an important new strategy to improve the efficacy of vaccines in
general, and that of Ad based vaccines specifically, by expression of SLAM system derived
adaptors simultaneous with antigen specific vaccinations. More specifically, in this study we
confirmed these notions by utilizing an Ad vaccine genetically engineered to express the SLAM
adaptor molecule EAT-2 along with the HIV derived antigen Gag. The inclusion of the AdEAT2 vector in Ad-HIV-gag vaccine cocktails significantly impacted upon the innate immune
responses induced by the vaccine cocktail, and more importantly specifically improved multiple,
antigen specific adaptive (cellular) immune responses to the target antigen present in the vaccine
cocktail, in this instance the HIV Gag protein.
There are a number of mechanisms as to how expression of EAT-2 might facilitate
induction of antigen specific immune responses in vivo. NK cells represent a subset of innate
immune cells that have been shown to play an important role in bridging innate and adaptive
immune responses, by influencing DC function (302), providing signals for augmenting Th1
immune responses (77, 78, 303), and inducing tumor-specific CTLs (304). In addition, it has
been shown recently that NK cell-mediated cytotoxicity of antigen-expressing target cells
induces robust antigen-specific adaptive immune responses (80). EAT-2 has been shown to be
indispensable in activating NK cell mediated cytotoxicity, by acting as a downstream adaptor
protein facilitating signaling from the SLAM family receptor CRACC (in mice) (151, 305) or
NTB-A (in humans) (306). Ad mediated transduction of the EAT-2 gene enhanced NK cell
activation (especially prolonged in those NK cells found in the circulation), activations that may
89
facilitate the activation and/or maturation of DCs thereby biasing the HIV-Gag specific immune
profile towards a Th1 response (78). Interestingly, we noted that Ads induced significantly
elevated levels of IFN-γ in NK cells regardless of EAT-2 overexpression. These results suggest
that EAT-2 overexpression enhances immune responses to the co-injected antigen in a
mechanism that does not involve induction of higher levels of IFN-γ producing NK cells. For
example, in addition to NK cells, we also observed increased activation of NKT cells after Ad
mediated transduction of the EAT-2 gene. Several reports have shown that enhancing the
activation of NKT cells can also positively influence the initial activation of DCs and/or NK
cells, thereby increasing DC-dependent adaptive (cellular) immune responses (85-89). Our data
suggests that harnessing this potential capability of NKT cells (in this instance via expression of
EAT-2) may be an important goal of “next generation" vaccines.
Previous reports have shown that SLAM derived signaling can also increase the antigen
presentation capabilities of APCs (291, 292). We note that EAT-2 is the only SLAM adaptor
molecule currently known to be expressed in APCs (290). These previous observations, suggest
that Ad mediated transduction of EAT-2 into DCs may have directly facilitated the induction of
antigen specific adaptive immune responses observed in this study. We confirmed that EAT-2
over-expression within several different types of APC triggers the induction of CD40, CD80,
CD86, and MHC class-II, all of which are critical co-stimulatory molecules that augment
subsequent APC activation of T cells. While it is known that Ads can transduce APCs and DCs
in vivo, other cell types may also be being transduced by the Ad vector expressing EAT-2, and
also be impacting upon our in vivo results. It is conceivable that EAT-2 overexpression also
facilitated SLAM - independent functions in Ad-EAT2 transduced immune cells. SAP family
adaptors can also interact, by way of their SH-2 domain, with other classes of receptors
90
-
expressed in immune cells including CD22 (expressed in both CD8α DCs and B cells) and
FcγRIIB (307, 308). Future studies will be required to delineate which aspects of the augmented
EAT-2 dependent immune responses may be mediated by these or other receptors.
Various studies in mouse models and non-human primates suggest that improving the
breadth of the antigen specific cellular immune responses elicited by a vaccine is positively
correlated with an improved ability of the vaccine to induce protective immunity for example
after pathogen challenge of vaccinated subjects (309, 310). Our results demonstrate that vaccine
cocktails possessing the ability to express EAT-2 along with a target antigen increased the
breadth of the cellular immune responses to the antigen, a result that correlated with a
significantly improved induction of antigen specific cytolytic T cell activity in vivo. We
confirmed these results in both C57BL/6 and Balb/c mice, (two mouse strains that can bias
adaptive immune responses to a Th1 or Th2 response, respectively) indicating that the "adjuvant"
effect of EAT-2 is not specifically limited by immuno-genetic background differences of the host
animal, at least in this species. In conclusion, our findings suggest that enhancing SLAM
signaling by expressing EAT-2 during antigen vaccination can serve to improve the ability of a
vaccine to stimulate the innate immune system, and subsequently induce improved, antigen
specific adaptive immune responses.
Acknowledgment:
We wish to thank Michigan State University Laboratory Animal support facilities for
their assistance in the humane care and maintenance of the animals utilized in this work. We
specifically thank, Dr. Sungjin Kim for his generous advice and suggestions. A.A. was
supported by the MSU Foundation, as well the Osteopathic Heritage Foundation. YAA was
91
supported by the King Abdullah bin Abdulaziz Scholarship, Ministry of Higher Education,
Kingdom of Saudi Arabia.
92
Chapter III
Vaccine platforms combining Circumsporozoite protein and potent immune modulators,
rEA or EAT-2, paradoxically result in opposing immune responses
This chapter is the edited version of a research article that was published in PLoS ONE Journal,
Volume 6, Issue 8 (e24147), August 30, 2011.
Authors: Aldhamen Y.A.*, Schuldt N.J.*, Appledorn D.M., Seregin S.S., Kousa Y., Godbehere
S., and Amalfitano. *these authors contributed equally to this work.
Authors’ contribution:
Aldhamen Y.A.*: Constructed the rAd5-EAT2 vaccine vectors, performed all the rAd5-CSP+
rAd5-EAT2 co-injection experiments [except the in vivo CTL assays], performed and analyzed
CSP-tetramer and ICS staining for rAd5-CSP+ rAd5-GFP/rEA, developed the protocol of in vivo
CTL assay and guide in performing the in vivo CTL testing for rAd5-CSP+ rAd5-EAT2 coinjection experiments, and gave some advice during manuscript writing.
Schuldt N.J. *: Performed all the rAd5-CSP+ rAd5-GFP/rEA co-injection experiments,
preformed in vivo CTL experiments for rAd5-CSP+ rAd5-EAT2 co-injection experiments,
performed all the ELISA analysis, and wrote the manuscript.
All other co-authors: Helped in performing experiment and participated in discussion during
manuscript writing.
93
3.1.
Introduction
In this chapter, I will provide a brief background about malaria and Plasmodium
parasites, the immune responses to malaria parasite infections, describe the current state of the
malaria vaccine field and outline strategies for the development of an effective malaria vaccine,
and finally introduce a novel strategy for developing a pre-erythrocytic-stage malaria vaccine by
utilizing adenoviral vectors that express the Plasmodium falciparum circumsporozoite protein
and the innate immune modulators rEA or EAT-2 proteins.
Malaria is an infectious disease that continues to devastate populations world-wide,
causing nearly 1 million deaths annually, and morbidity that overwhelms the medical capabilities
of developing countries. Malaria is caused by several species of the Plasmodia parasite that have
very complex life cycles involving a female Anopheles mosquito and a mammalian host. The
plasmodium parasite life cycle is a very complex multi-stage process including both obligate
intracellular asexual growth within mammalian hepatocytes (the pre-erythrocytic stages) and
erythrocytes (the blood or erythrocytic-stages) (311). Sexual differentiation, including the fusion
of gametocytes and parasite propagation, occurs in the mosquito vector (311). Five species of
Plasmodium are infectious for human: P. falciparum, P. vivax, P. malariae, P. ovale, and P.
knowlesi. Most cases of Malaria are caused by an infection with the protozoan parasite P.
falciparum. Plasmodium falciparum infections are responsible for about 80% of all malaria cases
and around 90% of all malaria deaths, therefore it has been the focus of most research (312).
However, recent studies in South East Asia have also shown that 25% of patients with severe
malaria also have P. vivax infection (313).
94
3.1.1
Immune responses to Plasmodium parasites
Similar to other microbial infections, infections by Plasmodium parasites have been
associated with enhanced activation of the innate immune system followed by adaptive immune
responses involving both B- and T-cells (314). Plasmodium parasite infections result in
activation of several innate immune cells including NK cells, NKT cells, DCs, Kuppfer cells, γδ
T cells, and macrophages (315). Several pro-inflammatory cytokines, such as TNFα, IL-1, IL-6,
IFNα and IFNγ, have been detected in responses to Plasmodium infection (316), thus stimulating
and regulate the adaptive immune responses. In addition, soluble products from Plasmodium
parasites have been shown to induce IFNα production from pDCs and to increase expression of
CD86 and CCR7 on pDCs in vitro (316). The induction of these pro-inflammatory cytokines has
been shown to require functional TLR (TLR2 and TLR9) and Myd88 signaling pathways (316,
317).
+
+
CD8 T cells and CD4 T cells have been shown to be activated by parasitic infection
(318). Studies in murine models and humans have demonstrated that T cells were important for
pre-erythrocytic (sporozoite-liver) stage immunity (319). In particular, the sporozoite’s major
+
surface protein, the Circumsporozoite (CS) protein, was shown to be a target for CD8 T cells
(320). T cell depletion studies prior to WT-sporozoite challenge in Balb/c mice have identified
+
CD8 T cells as the primary cytotoxic cells in radiated-sporozoite induced immunity (321). In
+
addition, it has been shown that after an infectious mosquito bite, CS Protein-specific CD8 T
cells, primed by skin-draining lymph nodes activated DCs, migrate to the liver and eliminate
+
parasite infected hepatocytes (322). Moreover, studies have also revealed that CD8 T cellderived IFNγ, but not the lytic factors (perforin, Granzyme B, or Fas), are the main effector
95
+
molecule that mediates protection following WT-sporozoite challenge (323). CD4 T cells have
also been shown to play a critical role during parasite infection in murine malaria models. For
+
example, immunization of β2-microglobulin deficient mice (which fail to induce CD8 T cell
responses) with either P. berghei or P yoelii radiated sporozoites, rendered these mice
completely protected from subsequent challenge (324). Similarly, studies utilizing genetically
+
+
attenuated whole parasite (GAPs) immunization strategies have also shown that CD4 , CD8 T
cells, and IFNγ are each key mediators of protective immunity (325). Gamma-delta T cells have
also been demonstrated to contribute to protection in P. yoelii radiation sporozoite-immunized
mice lacking conventional αβ T cells (326). In addition, regulatory T cells have also been shown
to play a role during plasmodium infection. Treg cells have been shown to associate with
increased IL-10 and TGFβ secretion, diminished pro-inflammatory cytokine production, and
decreased antigen-specific immune responses (327).
The role of B cell responses to malaria parasites has also been studied. During natural
infection, antibodies against both the merozoite surface proteins (MSPs) and erythrocyte
membrane-associated antigens (EMPs) have been detected. These antibodies were found to have
growth inhibitory properties in vitro including, inhibiting invasion of new erythrocytes, blocking
the sequestration of infected erythrocytes to endothelial cells, and promoting opsonization, thus
enhancing phagocytic activity of monocytes and macrophages (328). However, studies in both
humans and mice have shown that memory B cell responses are poorly induced or are short-lived
as a result of natural infection.
Despite these immune responses, malaria parasites have developed a variety of
mechanisms that allow them to evade immunity and persist in the host. Several studies have
96
shown that enhancing apoptosis of T cells and other effector cells (329), interfering with
presentation and processing (330), mutating the sequence of epitopes critical for T- or B-cell
recognition (331), and induction of T and B cell exhaustion (332), are all mechanisms that have
been described for immune evasion by Plasmodium parasites.
3.1.2
Malaria vaccine development
It is known that prophylactic vaccination of subjects with irradiated sporozoites can result
in 94% protection against subsequent malaria infection, a result confirming that induction of
protective immunity to malaria antigens can be achieved, if the vaccine utilized is potent enough
and expresses the correct antigenic targets (314, 333). Unfortunately, the large scale production
of irradiated sporozoites for this purpose has not been feasible, largely due to the difficulties
associated with cGMP production of this class of vaccine. Significant efforts have been
undertaken to create malaria specific subunit vaccines. Malaria subunit vaccines that target
blood-stage malaria infection with recombinant P. falciparum erythrocyte membrane protein-1
(PfEMP-1) or merozoite surface proteins have failed to achieve protection against malaria in
humans, despite production of strong antibody responses (334).
Some of the most successful malaria vaccine studies that target the liver-stage, to date,
have attempted to induce adaptive immune responses to the P. falciparum CS protein. CS protein
is an ~58 kDa surface protein composed of a middle repeat region consisting of multiple NANP
repeats that are flanked by a C-terminus containing a thrombospondin-like type I repeat region
(TSR) and an N terminal region that assists with liver cell attachment (Figure 1) (335).
Figure 16: CS protein sequence. The CS protein sequence utilized for constructing the Ad-CSP
vaccine was designed based on several known CS protein sequences. The NYDNAGTNL
97
peptide’s location is underlined in the sequence. Bold font within the sequence indicates the
repeat region of CS protein. The location of the Thrombospondin-like Type 1 repeat region (TSR
domain) is indicated by gray font.
98
Figure 16: CS protein sequence.
99
CS protein is expressed on the surface of sporozoites and is also expressed in the plasma
membrane and cytoplasm of infected hepatocytes during early liver infection (336). Induction of
potent cellular immune responses to CS protein by a prophylactic malaria vaccine could
potentially eradicate both sporozoites and infected hepatocytes, potentially stopping the infection
before clinical symptoms occur.
+
Multiple studies have demonstrated the importance of CD8 T cell responses in
combating murine malaria infections (337-340). An oral salmonella vaccine expressing murine
malaria derived CS protein was capable of protecting antibody deficient animals (339). In
+
addition, passive transfer of CD8 T cells that recognize a specific murine malaria CS protein
antigen resulted in 100% survival upon sporozoite challenge (338). Ads expressing murine
malaria derived CS protein have been shown to be capable of providing cytotoxic T cell
mediated inhibition of parasite liver stage development up to 93% (340). One of the most
successful subunit malaria vaccines to date contain a hepatitis B viral surface protein (HBsAg)
CS fusion protein (referred to as RTS,S). Initial formulations of the RTS,S based vaccines
demonstrated little protection. Subsequent trials combining several novel “AS” series adjuvants,
(the latter consisting of different preparations of monophosphoryl lipid A (MPL) and a plant
extract known as QS21), with the HBsAg-CSP fusion protein (referred to as RTS,S/ASO1B)
improved induction of CS specific responses, but this level of protection was still limited,
suggesting that a more robust CS protein specific immune response may be required to achieve
improved protection rates (341-347).
Recombinant adenoviruses (rAd) can be utilized to induce potent adaptive immune
responses to antigens that they are genetically engineered to express. For example, rAd serotypes
5 and 35 expressing CS protein (Ad5.CS and Ad35.CS, respectively) are capable of inducing CS
100
protein specific T and B cells in mice similar to the levels induced by the RTS,S/ASO1B
adjuvanted vaccine (348, 349). These levels paralleled results achieved in mice treated with a
rAd35 vaccine expressing the P. Yoelli derived CS protein, a vaccine that was also found to be
capable of providing up to a 92-94% inhibition of liver infection when vaccinated animals were
challenged intravenously (IV) with viable P. yoelli sporozoites (350). Combining rAd35CS and
rAd5CS CS protein expressing vaccines in a heterologous prime-boost regimen in rhesus
monkeys can induce a more potent T and B cell response than use of either vaccine alone (351).
Heterologous prime-boost vaccination regimens utilizing Ad35.CS protein and RTS,S/ASO1B
have also been analyzed and show significant improvement over either vaccine platform alone
(352). However, the utilization of alternative serotype rAds or chimp derived rAds as a vaccine
platform may be dangerous, as our studies and those of others have confirmed the increased
innate toxicity of non-Ad5 rAds (353). Therefore, improving the capability of rAd5 vaccines to
induce more potent antigen specific adaptive immune responses is a high priority in the drive to
find an efficacious malaria vaccine. In this study we sought to improve CS protein specific cell
mediated immune (CMI) responses induced by CS protein-expressing rAd5s by co-expression of
innate immune response modulating proteins by the vaccine platform.
The innate immune system plays an integral role in augmenting and/or shaping the
induction of antigen specific adaptive immune responses (2). A group of cellular receptors that
recognize a variety of pathogen derived antigens, known as the toll-like receptors (TLRs), play a
crucial role in identifying PAMPs, and then augmenting adaptive responses to those PAMPs. We
have previously confirmed that rAds ability to induce innate and adaptive responses are
dependent upon several TLR’s, and that many of these responses are primarily dependent upon
MyD88 functionality (179, 184). We have also recently demonstrated that when rAd5 vaccines
101
engineered to express a novel Eimeria tenella derived TLR agonist, rEA, are co-administered
with rAd5 vaccines expressing a target antigen there was significant improvement in the ability
of the vaccine to induce antigen specific cellular immune responses (133, 354, 355). Similarly,
we have recently confirmed that co-expression of adaptor proteins derived from SLAM receptors
signaling pathways can also augment induction of beneficial immune responses to rAd expressed
antigens (191, 356). In the latter instance we utilized the SLAM family of receptors adaptor
protein (EAT-2), an adaptor protein known to mediate SLAM receptor signaling in innate
immune cells (357, 358).
In this study, we determined what the impact of modulation of innate immune responses
during CS protein presentation would have upon induction of subsequent CS protein specific
immune responses in vivo. Unexpectedly, use of a TLR agonist uncovered a potent
immunosuppressive activity inherent to the combined use of rEA and CS protein, an activity that
mitigated induction of any CS protein specific adaptive immune responses. Fortunately,
expression of the SLAM receptors adaptor protein EAT-2 overcame and enlightened possible
mechanisms underlying the paradoxical CS protein immunosuppressive activity we uncovered
when stimulating TLR pathways.
102
3.2
Results
CS protein expressed from rAd5 based vaccines can induce CS protein specific B and T
cell responses. A rAd5 based vaccine expressing a codon optimized form of the CS protein (AdCSP) was constructed as shown in figure 17.
Figure 17: Ad-CSP construction. Recombinant Ad-CSP was constructed by creating a codon
optimized CS protein sequence flanked by NheI sites in a pGA4 plasmid. The sequence was
excised with the Nhe1 and cloned into a pShuttle containing a CMV expression cassette. The
resulting plasmid was linearized with PmeI and recombined with pAdeasyI Ad5 vector in BJ
5183 cells. pAd-CSP was then purified and linearized with PacI enzyme and transfected into
HEK 293 cells from which Ad-CSP was purified using cesium gradients.
103
Figure 17: Ad-CSP construction.
104
A dose response study was initially performed to assess at what dose optimal CS specific
B and T cell responses could be detected. BALB/cJ mice were intra-muscularly (IM) injected
7
9
with varying doses of Ad-CSP ranging from 1×10 to 1×10 virus particles (vps) per mouse. At
14 days post injection (dpi), splenocytes derived from the vaccinated mice were harvested and
exposed to an immunodominant CS protein derived peptide (NYDNAGTNL). Significantly
increased numbers of IFNγ secreting splenocytes were noted in Ad-CSP vaccinated mice treated
7
9
8
with 5.0×10 to 1.0×10 vps, with peak numbers achieved at a dose of 1.0×10 vps/mouse.
Higher Ad-CSP doses resulted in a trend of decreasing, though not significantly less, numbers of
spot forming cells (SFCs) (Figure 18A). Interestingly this phenomenon has also been observed
by other groups; however, an explanation for this phenomenon has yet to be provided (359, 360).
These finding were further supported in individual splenocytes derived from the vaccinated
+
mice, where CD8 T cell IFNγ, TNFα, and IL-2 levels were measured by intracellular staining
8
(ICS) using flow cytometry. IFNγ and TNFα production peaked at the 5.0 ×10 vps/mouse with
9
similar decreasing trend occurring at 1.0 ×10 vps/mouse. IL-2 producing cells were much lower
7
in percentage, with the greatest numbers being observed as the 5.0×10 vps/mouse dose (Figure
18B). We will further discuss the importance of these findings in the discussion section.
To determine if Ad-CSP is also capable of stimulating B cell responses specific to the CS
protein, plasma was collected from the vaccinated mice and assayed by an IgG CS protein
specific ELISA at 14 dpi. Significant increases in CS specific IgG were detected in all mice
7
treated with Ad-CSP with a peak response occurring at 5.0×10 vps/mouse, demonstrating Ad-
105
CSP is capable of stimulating a B cell response against CS protein even at the lowest dose used
in the study (Figure 18C).
Figure 18: Ad-CSP Stimulates CS protein specific T and B cell responses. CS protein
specific immune responses increase in an Ad-CSP dose dependent manner. BALB/cJ mice (n=3)
7
9
were injected IM with Ad-CSP ranging from 1x10 to 1x10 vps/mouse, increasing by half logs.
14 days post injection splenocytes and plasma were collected. (A) ELISpot assays were
performed to quantify IFNγ secreting cells from splenocytes stimulated with CS protein peptide,
NYDNAGTNL, ex vivo. (B) IFNγ, TNFα, and IL-2 expression by splenocyte derived CD3
+
+
CD8 T cells was analyzed by flow cytometry following ex vivo stimulation with
NYDNAGTNL. (C) Total IgG against CS protein was assessed by ELISA. The bars represent
mean ± SD. Statistical analysis was completed using One Way ANOVA with a StudentNewman-Keuls post-hoc test, *,**,*** denotes significance over naïve, p<0.05, p<0.01,
p<0.001.
106
Figure 18: Ad-CSP Stimulates CS protein specific T and B cell responses.
107
Previous experiments confirm that expressing the TLR agonist rEA from an Ad vector
stimulates innate immune responses during Ad-mediated vaccination, responses that positively
correlated with improved induction of antigen specific adaptive immune responses against
several antigens, such as the HIV antigen, Gag (133). In this study, we sought to utilize rEA to
improve induction of CS protein specific immune responses. We first confirmed that expression
of rEA along with CS protein facilitated induction of pro-inflammatory innate immune
responses, responses we had noted in our previous studies of rEA. Plasma cytokine levels at 6
10
hours post injection (hpi) in mice co-injected intravenously (IV) with either 3.75×10
10
Ad-CSP and 3.75×10
vps of
vps Ad-GFP/rEA were compared to responses measured after identical
co-injections utilizing an Ad-GFP expressing vector (that does not express rEA) as a control
(Figure 17) . We observed significantly higher levels of IL-6, IL-12(p40), G-CSF, MCP-1, MIP1β, RANTES, KC, and TNFα in mice treated with Ad-CSP +Ad-GFP/rEA as compared to
control virus treated animals, as well as, mock infected animals (Figure 19).
Figure 19: TLR agonist, rEA, induced innate cytokines 6 hours post injection. Co-injection
of Ad-GFP/rEA and Ad-CSP stimulated robust expression of innate cytokines and chemokines
10
as compared to the control vaccine. BALB/cJ mice were injected IV with either 3.75×10
10
vps/mouse of Ad-CSP +Ad-GFP or 3.75×10
vps/mouse Ad-GFP/rEA +Ad-CSP. Plasma was
collected at 6 hours post injection. Plasma cytokine/chemokine levels were measured with a
mouse multiplexed bead array based quantitative system. The bars represent mean ± SD.
Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls posthoc test, *,** denotes significance between treatments, p<0.05, p<0.01.
108
Figure 19: TLR agonist, rEA, induced innate cytokines 6 hours post injection.
109
To assess the impact that these early increases in cytokine and chemokine responses had
7
on cell mediated immune (CMI) responses to CS protein we IM co-injected 5×10 vps/mouse of
7
Ad-CSP and 5×10 vps/mouse of Ad-GFP/rEA and compared the induction of CS specific
7
adaptive immune responses to those noted in our control mice receiving 5×10 vps/mouse of Ad7
CSP and 5×10 vps/mouse of Ad-GFP IM, or mock infected mice. Splenocytes derived from
mock vaccinated mice did not show the presence of CS protein specific CMI responses while
Ad-CSP +Ad-GFP confirmed induction of CS protein specific CMI responses using ELISpot
analysis (p<0.05) (Figure 20A). However, despite the rEA enhanced activation of the innate
immune responses noted in Figure 19, ELISpot analysis of splenocytes derived from Ad-CSP+
Ad-GFP/rEA vaccinated animals confirmed a profound lack of induction of average CS protein
specific CMI responses, responses that were essentially identical to CS responses measured in
naïve mice (p>0.05) (Figure 20A).
Figure 20: Immuno-modulating proteins conversely affect IFNγ secreting splenocytes.
Co-vaccination with Ad-CSP and Ad-EAT2 dramatically increases IFNγ secreting splenocytes in
response to stimulation with CS protein epitope, NYDNAGTNL. BALB/cJ mice were injected
7
7
7
IM with either 5×10 vps/mouse of Ad-CSP and 5×10 vps/mouse Ad-GFP or 5×10 vps/mouse
7
of Ad-CSP and 5×10 vps/mouse of either (A) Ad-GFP/rEA (n=5) or (B) Ad-EAT2 (n=6).
Splenocytes were collected 14 days post co-injection. ELISpot were performed on the
splenocytes of these mice stimulated with NYDNAGTNL peptide to assess the amount of IFNγ
secreting cells. The bars represent mean ± SD. Statistical analysis was completed using One Way
110
ANOVA with a Student-Newman-Keuls post-hoc test,* Denotes significance over naïve animals,
p<0.05. Representative figures of two independent experiments.
Figure 20: Immuno-modulating proteins conversely affect IFNγ secreting splenocytes.
111
Previously we have not observed an ablation of CMI responses when CS protein was coadministered with Ads expressing other antigens at these low doses, further suggesting that this
effect may be specific to simultaneous TLR stimulation (Figure 21).
Figure 21: CS protein expression does not interfere with antigen specific immune responses
against other transgenes at low doses. Co-vaccination with Ad-Gag +Ad-CSP did not result in
5
decreased gag specific immune responses. BALB/cJ mice were injected with 5×10 vp/mouse of
7
5
7
Ad-Gag and 5x10 vp/mouse of Ad-CSP or 5×10 vp/mouse of Ad-gag and 5×10 vp/mouse of
Ad-GFP. Splenocytes were collected 14 dpi and assayed by ELISpot for CS protein peptide
(NYDNAGTNL) specific IFNγ secretion (A) or gag peptide (AMQMLKETI) specific IFNγ
secretion (B). The bars represent mean ± SD. Statistical analysis for Supplemental Figure 3A
included other peptides tested from the peptide library that are not displayed in the graph. Two
Way ANOVA with Student-Newman-Keuls post-hoc test (A) or One Way ANOVA with a
Student-Newman-Keuls post-hoc test (B) were utilized for statistical analysis. **, *** denotes
significance between treatments, p<0.01, p<0.001.
112
Figure 21: CS protein expression does not interfere with antigen specific immune responses
against other transgenes at low doses.
113
Despite there being no significant differences between CS protein responses in Ad-CSP
+Ad-GFP/rEA treated animals and naïve animals, we did note that in one Ad-CSP +AdGFP/rEA animal there was some evidence of an elevated CS protein specific response,
independently verifying that this group did in fact receive viable Ad-CSP vector (Figure 20A).
Based upon the loss of CS protein responsiveness after utilizing TLR-mediated augmentation
along with CS protein antigen vaccination we hypothesized that the CS protein may have an
ability to mitigate induction of beneficial innate immune responses in the context of excessive,
TLR pathway mediated activation as the ablated immune responses were only observed after Ad6
GFP/rEA doses exceeded 5×10 vp/mouse (Figure 22).
7
Figure 22: Ad-GFP/rEA combined with 5x10 vp/mouse of Ad-CSP begins to display a
6
diminished CS protein specific CMI response after a dose of 5×10 vp/mouse. Only after the
6
dose of Ad-GFP/rEA exceeds 5×10 vp/mouse do we observe a diminished CS specific CMI
6
response when combined with 5x10 vp/mouse of Ad-CSP. BALB/cJ mice were injected with
6
8
7
doses ranging from 5×10 to 5x10 vp/mouse of Ad-GFP/rEA combined with 5x10 vp/mouse
of Ad-CSP. Splenocytes were collected 14 dpi and were analyzed by flow cytometry for
+
+
+
NYDNAGTNL tetramer CD3 and CD8 cells (A) or ELISpot for CS protein specific IFNγ
secretion (B). Statistical analysis was completed using One Way ANOVA with a StudentNewman-Keuls post-hoc test, *** denotes significance between treatments, p<0.01, p<0.001.
114
7
Figure 22: Ad-GFP/rEA combined with 5x10 vp/mouse of Ad-CSP begins to display a
6
diminished CS protein specific CMI response after a dose of 5×10 vp/mouse.
115
To attempt to test this hypothesis, we made use of a recently described, alternative
method for augmenting induction of antigen specific adaptive immune responses, utilizing Ad
mediated co-expression of a SLAM receptor signaling pathway adaptor, EAT-2, along with a
7
targeted antigen (191, 356). To accomplish this we co-injected 5×10 vps/mouse of Ad-CSP and
7
5×10 vps/mouse of Ad-EAT2, and compared the induction of CS specific adaptive immune
7
7
responses to those noted in the control mice receiving 5×10 vps/mouse of Ad-CSP and 5×10
vps/mouse of Ad-GFP IM, as well as mock vaccinated mice. Again, splenocytes were collected
at 14 dpi and stimulated with the CS derived peptide NYDNAGTNL ex vivo. In dramatic
contrast to our previous results utilizing the Ad-GFP/rEA and Ad-CSP vaccination strategy,
splenocytes from mice co-treated with Ad-CSP and Ad-EAT2 had significantly more IFNγ
secreting cells than splenocytes from both mock injected mice as well as mice co-treated with the
control vaccine (p<0.05) (Figure 20B). Given these results, we sought to further characterize the
EAT-2 dependent improvement in CS specific immune responses by flow cytometry. Peripheral
blood mononuclear cells (PBMC) derived from the vaccinated animals were stained with CD3
and CD8 fluorescent antibodies, as well as a NYDNAGTNL peptide loaded tetramer. AdCSP+Ad-EAT2 treated mice had significantly higher percentages of CS protein specific tetramer
positive CD8+cells present in their PBMCs than the percentage noted in the Ad-CSP +Ad-GFP
+
+
control group (p<0.001) (Figure 23A). CD3 CD8 splenocytes were additionally analyzed for
+
+
IFNγ and perforin by ICS using flow cytometry. The percent of CD3 CD8 cells that secreted
IFNγ was significantly higher in Ad-CSP+Ad-EAT2 treated mice as compared to Ad-CSP +AdGFP treated control (p<0.05) (Figure 23B). The percent of CS protein peptide specific CD3
116
+
+
+
CD8 perforin cells also tended to be higher in animals given the Ad-EAT2+Ad-CSP
vaccination cocktails however this did not reach statistical significance (Figure 23C).
Figure 23: Co-expression of CS protein and EAT-2 stimulates more potent CS protein
specific CMI responses. Co-vaccination with Ad-CSP and Ad-EAT2 resulted in increased
+
+
NYDNAGTNL tetramer positive CD8 T cells as well as improved IFNγ secretion from CD8 T
7
cells. BALB/cJ mice (n=6) were co-injected IM with 5x10 vps/mouse of Ad-CSP and 5x10
7
7
7
vps/mouse of Ad-EAT2 or 5x10 vps/mouse of Ad-CSP and 5x10 vps/mouse of Ad-GFP. (A)
Peripheral Blood Mononuclear Cells (PBMCs) were stained with CD8-Alexa Flour700, CD3APC-Cy7, and CSP (NYD)-Tetramer. (B-C) Intracellular staining was performed on splenocytes
after stimulation with NYDNAGTNL peptide. Cells were stained with CD8-Alexa Flour700,
CD3-APC-Cy7, ViViD, IFNg-APC, and Perforin-PE antibodies. The bars represent mean ± SD.
Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls posthoc test, *, **, *** denotes significance over naïve animals, p<0.05, p<0. 01, p<0.001
117
Figure 23: Co-expression of CS protein and EAT-2 stimulates more potent CS protein
specific CMI responses.
118
To confirm that the differences in the responses observed are not a result of GFP antigens
competing with CS protein antigens, but are in fact a direct result of the expression of EAT-2 we
injected mice with Ad-CSP +Ad-GFP or Ad-CSP + an empty Ad vector (Ad-Null). We observed
no differences between the treatments, indicating GFP does not interfere with induction of CS
protein specific CMI responses (Figure 24).
Figure 24: Expression of GFP does not interfere with CS protein specific CMI responses.
Co-injection of Ad-GFP does not interfere with Ad-CSP initiated CS protein specific CMI
7
responses. BALB/cJ mice were co-injected with 5×10 vp/mouse of Ad-GFP and 5x10
7
7
7
vp/mouse of Ad-CSP or 5×10 vp/mouse of Ad-Null and 5x10 vp/mouse of Ad-CSP.
+
+
+
Splenocytes were collected 14 dpi and cells were measured for NYDNAGTNL tet , CD3 CD8
+
+
+
T-cells. Both treatments had a higher percentage of CS protein specific tet , CD3 CD8 T-cells
than Naïve with no difference observed between Ad-CSP + Ad-Null and Ad-CSP +Ad-GFP.
Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls posthoc test, *** denotes significance between treatments, p<0.01, p<0.001
119
Figure 24: Expression of GFP does not interfere with CS protein specific CMI
responses.
120
Increased breadth of CMI responses to a pathogen derived protein has been shown to be
beneficial relative to eventual protection against actual pathogen challenge (262, 309, 361). To
detect CMI responses against other peptides present within the CS protein (and therefore to
gauge the breadth of response against the whole CS protein) we generated a CS protein specific
peptide library. This library consists of 15 mer peptides that overlap each other by 5 amino acids
and spans the non-repeating regions of the full length CS protein. At 14 dpi, pooled splenocytes
derived from the control or experimental groups of vaccinated animals were stimulated ex vivo
with one 15mer peptide per well. Mice co-vaccinated with Ad-CSP and Ad-GFP/rEA had an
overall lower breadth of response as is evident by the number of wells with more than 15 spots
(Figure 25A). In contrast to the response seen in rEA treated animals, animals co-vaccinated with
Ad-CSP and Ad-EAT2 demonstrated a dramatic increase in breadth of response to CS derived
peptides when similarly analyzed (Figure 25B).
Figure 25: Co-expression of CS protein and EAT-2 increases the breadth of response
against CS protein. Increased breadth of response against CS protein epitopes was observed in
mice co-vaccinated with Ad-CSP and Ad-EAT2 as compared to the control vaccine. Splenocytes
from groups of five BALB/cJ mice were collected and pooled together from 14 days post
injection with either innate modulating treatments or control. ELISpots were performed to
measure IFNγ secreting cells when stimulated with a CS protein peptide library made up of 52
15mers that overlap by 5 aa on either side. Wells that contained more than 15 spots were counted
and compared between treatment groups (inset). (A) Mice were co-injected IM with 5x107
7
7
vps/mouse of Ad-CSP and 5x10 vps/mouse of Ad-GFP/rEA or 5×10 vps/mouse of Ad-CSP
7
7
and 5×10 vps/mouse of Ad-GFP. (B) Mice were co-injected IM with 5x10 vps/mouse of Ad7
7
7
CSP and 5×10 vps/mouse of Ad-EAT2 or 5x10 vps/mouse of Ad-CSP and 5×10 vps/mouse
121
of Ad-GFP. As a negative control, naïve splenocytes were also tested against paired peptides
from the peptide pool, with an average background of spots per paired peptides being only 2.2
spots.
122
Figure 25: Co-expression of CS protein and EAT-2 increases the breadth of response
against CS protein.
123
To better assess the functional consequence of the improved CS specific CMI responses
noted by expression of EAT-2, we stimulated splenocytes from naïve mice, mice vaccinated with
the control vaccine, and mice vaccinated with Ad-CSP +Ad-EAT2 with NYDNAGTNL ex vivo,
+
+
then analyzed them by flow cytometry for CD3 , CD8 T cells that were also positive for a
degranulation marker, CD107a. Both control treated and Ad-CSP +Ad-EAT2 treated mice
+
+
demonstrated a significantly higher number of CD8 , CD107a T cells than those quantified in
+
naïve mice, indicating increased ability of CD8 T cells to express granules when stimulated
with a CS protein epitope (Figure 26). However, the assay was not sensitive enough to measure a
difference between the control vaccinated mice and Ad-CSP+Ad-EAT2 vaccinated mice.
Figure 26: Improved degranulation of CD8+ T cells in mice co-vaccinated with Ad-CSP
and Ad-EAT2. Degranulation marker, CD107a, expression in CD8+ T cell from mice covaccinated with Ad-CSP+Ad-EAT2 or Ad-CSP +Ad-GFP. Splenocytes were collected from
7
7
BALB/cJ mice 14 days post co-injection of either 5x10 vps of Ad-CSP and 5x10 vps of Ad7
7
6
GFP or 5x10 vps of Ad-CSP and 5x10 vps of Ad-EAT2. 2×10 splenocytes from naive or
mice co-vaccinated with either treatment were stimulated with 2ug NYD-peptide at 37°C for 3
days. Cells were then washed with FACS buffer and stained with CD8-Alexa700, CD107-FITC
+
+
antibodies and viability dye (ViViD) and ran on LSR-II. % of live CD107 CD3 T cells is
shown. The bars represent mean ± SD. Statistical analysis was completed using One Way
ANOVA with a Student-Newman-Keuls post-hoc test,* Indicates significance over naïve p<0.05
124
Figure 26: Improved degranulation of CD8+ T cells in mice co-vaccinated with Ad-CSP
and Ad-EAT2.
125
We then conducted a more sensitive in vivo CTL assay (362). Mice were co-vaccinated
8
8
8
with either 1x10 vp/mouse of Ad-CSP and 1x10 vps/mouse of Ad-GFP or 1x10 vp/mouse of
8
Ad-CSP and 1x10 vps/mouse of Ad-EAT2. 14 days later vaccinated mice were treated with
CFSE labeled splenocytes that had been incubated with either the NYDNAGTNL peptide, or a
non-specific control peptide, and the elimination of NYDNAGTNL pulsed cells (CFSE
high
cells) was measured by flow cytometry. Based on the calculated percent specific killing, mice
vaccinated with Ad-CSP+Ad-EAT2 were more effective at killing cells exposed to the
NYDNAGTNL peptide than mice vaccinated with the control Ad-CSP vaccine (Figure 27).
Figure 27: Co-expression of CS protein and EAT-2 increases cytolytic activity of CS
protein specific T cells. Co-vaccination of mice with Ad-CSP and Ad-EAT2 increased specific
killing cells pulsed with CS protein peptides. BALB/cJ mice (n=4) were co-injected IM with
8
8
8
either 1x10 vps/mouse Ad-CSP and 1x10 vps/mouse Ad-GFP or 1x10 vps/mouse Ad-CSP
8
and 1x10 vps/mouse Ad-EAT2 on Day 0. Day 14 splenocytes were collected from naïve mice
and pulsed with either NYDNAGTNL peptide or an irrelevant peptide. NYDNAGTNL pulsed
splenocytes were stained with a high concentration of CFSE while splenocytes pulsed with
irrelevant peptide were stained with a low concentration of CFSE. Stained splenocytes were then
combined in equivalent doses. 8 million cells were then injected IV into naïve, Ad-CSP + AdGFP co-vaccinated, or Ad-CSP+Ad-EAT2 co-vaccinated mice. After 18 hrs splenocytes from
these mice were collected and analyzed by flow cytometry to assess the amount of
NYDNAGTNL specific killing.
126
Figure 27: Co-expression of CS protein and EAT-2 increases cytolytic activity of CS
protein specific T cells.
127
CS protein antibody specific ELISAs were also performed on plasma derived from AdCSP +Ad-GFP/rEA and Ad-CSP +Ad-GFP treated mice. CS protein specific total IgG antibody
levels in control vaccine treated animals were significantly elevated (p<0.05) as compared to
naïve animals. However, there was again no significant difference observed in Ad-CSP +AdGFP/rEA treated animals when compared to naïve animals (p<0.05) (Figure 28A). Conversely,
plasma collected from Ad-CSP+Ad-EAT2 treated animals had significantly higher levels of CS
protein specific IgG as compared to levels detected in naïve mice (p<0.05) (Figure 28B).
However, the mice receiving the control vaccine treatment had higher total CS protein specific
IgG levels than naïve and Ad-CSP +Ad-EAT2 treated animals (p<0.05) (Figure 28B).
Figure 28: Induction of CS protein specific antibody responses by Ad-CSP vaccines
augmented by rEA or EAT-2. Total IgG antibody against CS protein is ablated in Ad-CSP
+Ad-GFP/rEA co-vaccinated mice while Ad-CSP+Ad-EAT2 co-vaccinated mice demonstrated
significantly more CS protein specific IgG than naïve animals. BALB/cJ mice (n=5) were co7
injected IM with 5x10 vps/mouse of Ad-CSP and 5x107 vps/mouse of Ad-GFP or 5x10
7
7
vps/mouse of Ad-CSP and 5x10 vps/mouse of either (A) Ad-GFP/rEA or (B) Ad-EAT2. Plasma
was collected at day 14. Total IgG against CS protein in the plasma was measured by ELISA.
The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with
a Student-Newman-Keuls post-hoc test, * Denotes significance over naïve p<0.05. † Denotes
significant difference between treatments p<0.05.
128
Figure 28: Induction of CS protein specific antibody responses by Ad-CSP vaccines
augmented by rEA or EAT-2.
129
Further isotyping of IgG was performed, the ratios of Th1 to Th2 antibody (IgG2a/IgG1)
in mice treated with Ad-EAT2 +Ad-CSP were similar to the ratio of Th1 to Th2 antibody in
control treated mice in all dilution except 1:400, indicating that expression of EAT-2 did not
induce a Th1 to Th2 bias in these mice at 14 dpi as measured by this assay (Figure 29).
Figure 29: Sub-isotype analysis of IgG antibody from plasma of mice co-vaccinated with
7
Ad-CSP and Ad-EAT2. BALB/cJ mice (n=6) were co-injected i.m. with 5x10 vps of Ad-CSP
7
7
7
and 5x10 vps of Ad-GFP or 5x10 vps of Ad-CSP and 5x10 vps of Ad-EAT2. Plasma was
collected at day 14. (A) The amount of CS protein specific IgG sub-isotypes was measured by
ELISA. (B) The ratio of IgG2a/IgG1 was calculated to indicate the Th1 to Th2 response ratio.
The bars represent mean ± SD. Statistical analysis for sub-isotyping (A) was completed using
One Way ANOVA with a Student-Newman-Keuls post-hoc test and standard t-test was
performed for Th1 to Th2 ratios (B),* indicates significance over naïve p<0.05. † Indicates
significance between treatments p<0.05.
130
Figure 29: Sub-isotype analysis of IgG antibody from plasma of mice co-vaccinated with
Ad-CSP and Ad-EAT2.
131
In addition, when measured by intracellular staining, there was no significant difference
+
-
+
in the number of likely CD4 IFNγ expressing T-cells, as the number of CD8 CD3 T cells in
Ad-CSP +Ad-EAT2 treated animals and were similar to the numbers of these cells noted in AdCSP +Ad-GFP treated animals (Figure 30).
+
-
+
Figure 30: CD3 CD8 IFNγ cells respond similarly to both vaccine regimens. Co+
-
vaccination with Ad-CSP and Ad-EAT2 resulted in similar IFNγ secretion from CD3 CD8 T
7
cells. BALB/cJ mice (n=6) were co-injected IM with 5x10 vps/mouse of Ad-CSP and 5x10
7
7
7
vps/mouse of Ad-EAT2 or 5x10 vps/mouse of Ad-CSP and 5x10 vps/mouse of Ad-GFP.
Splenocytes were stimulation with NYDNAGTNL peptide. Cells were stained with CD8-Alexa
Flour700, CD3-APC-Cy7, ViViD, and IFNγ-APC. The bars represent mean ± SD. Statistical
analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test.
132
+
-
+
Figure 30: CD3 CD8 IFNγ cells respond similarly to both vaccine regimens.
133
3.3.
Discussion:
Our earlier works and those of others suggest that activation of the innate immune system
can play an important role in beneficially augmenting subsequent antigen specific adaptive
immune responses (179, 184, 363-365). For example, we previously augmented CMI responses
against HIV-Gag by co-injecting a rAd5 vector expressing HIV-Gag with a rAd5 vector
expressing a TLR agonist, rEA (363). Similarly, co-injecting a rAd5 vector expressing HIV-Gag
with a rAd5 vector expressing the SLAM receptors adaptor protein EAT-2 also augmented
induction of innate immune responses, and improved the induction of HIV-Gag specific T cell
responses (358). As a new approach to increasing the potency of malaria specific vaccines, we
now describe the use of adenoviral based vaccines engineered to express malaria derived
proteins, simultaneously administered with rAds expressing proteins known to modulate the
innate immune system. Most importantly, we have confirmed that rAd mediated expression of a
SLAM pathway derived adaptor (EAT-2) can significantly augment the induction of malaria
antigen (CS protein) specific CMI responses. This was verified based upon ELISpost analysis of
splenocytes (both as to their responsiveness to immunodominant peptides, as well the breadth of
these responses to the full length CS protein), ICS staining of cells for IFNγ, and most
importantly by a CS protein specific in vivo CTL functional assay. EAT-2 expressing vaccines
should be considered for use in future malaria vaccine trials attempting to boost malaria antigen
specific CMI responses. Furthermore, EAT-2 co-expression allowed for the induction of CS
specific antibody responses as well.
In contrast, co-vaccination of mice with a rAd vaccine expressing a TLR agonist
simultaneously with a rAd expressing CS protein, actually had the opposite effect, and
completely mitigated induction of CS protein specific adaptive humoral and cellular immune
134
responses, as compared to responses typically induced by the rAd vaccines expressing CS
protein alone. There could be numerous reasons for these unexpected, paradoxical and
potentially disturbing results. A simple reason could be that the increase in pro-inflammatory
cytokines caused by rAd mediated expression of the TLR agonist, rEA, could be influencing
expression of CS protein from the rAd5 vector. However, this effect would have likely been
observed in our previous studies utilizing the same vector combinations, as well as the same TLR
or SLAM receptors derived adaptors, but a different target antigen (HIV-Gag). Those studies
also confirmed induction of similar innate immune responses to those noted in this study (358,
363). It is more logical that the CS protein somehow negatively interacts with immune pathways
excessively activated by TLR agonists such as rEA, resulting in a complete ablation of CS
protein specific CMI responses. This immunosuppressive activity of CS protein appears to only
be unveiled after excessive stimulation of TLR pathways, as our use of EAT-2 demonstrated not
only avoidance of CS immunosuppressive activity, but also allowed for enhanced induction of
CS specific adaptive immune responses.
The CS protein has been specifically confirmed to be capable of outcompeting the
transcription factor NF-κB for binding to the nuclear transport protein, importan α, resulting in
the down-regulation of at least forty NF-κB controlled genes (366). CS protein was also shown
to inhibit NF-κB entry into the nucleus by 75% (366). As NF-κB is known to control numerous
genes involved in pro-inflammatory immune responses, one hypothesis may be that the CS
protein can downregulate excessive (TLR-driven) NF-κB transcriptome responses, and result in
a dramatically diminished acute inflammatory response, thereby blunting subsequent CS protein
antigen specific adaptive immune responses (367). This may make biological sense, as infection
of hepatocytes by malaria sporozoites has been shown to induce the activation of NF-κB in a
135
MyD88 specific manner (368). Expression of CS protein by the parasite may have evolved to
counteract this inflammatory response and prevent excessive induction of malaria specific
adaptive immune responses in the infected host. Interestingly, recent studies on an
immunosuppressive drug (dehydroxymethylepoxyquinomicin) that specifically interferes with
the NF-κB-importan α interaction was shown at lower doses to only modestly affect IL-6 and
TNFα levels, while dramatically affecting Th1 expansion, results paralleling those noted in our
experiments (369).
These notions may also explain our findings, as well results previously reported by others
(348, 359). Those studies and ours verify that at very high doses, rAd vaccines expressing CS
protein also show a trend toward diminished induction of CS protein specific CMI responses
(Figure 18) (348, 359). Multiple studies have shown that Ad vectors can also induce NF-κB
(181, 370). Quite possibly, the CS protein immunosuppressive effects are not uncovered until an
“NF-κB activation threshold” has been broached, in this instance by use of excessively high
doses of rAd vaccines expressing CS protein, or by using more modest doses of the Ad vaccine
coupled with potent TLR activation. Further studies will need to be performed to elucidate
whether this or other mechanisms may be responsible for our results. Regardless, our data
demonstrate the need to consider the impact the inclusion of CS protein derived peptides, or the
entire protein along with other immunostimulatory compounds may have upon present and future
malaria specific vaccines. Taken together with recent data demonstrating that protection from
malaria challenge can be independent of CS protein suggests that the use of CS protein in certain
malaria vaccine formulations will have to be carefully considered (371, 372).
In contrast to co-expression of the TLR agonist, co-expression of EAT-2 and CS protein
eventuated in the enhanced induction of CMI responses to the CS protein, relative to the use of
136
the Ad-CSP vaccine alone. We have also previously observed a potent CMI response against
HIV derived Gag in mice treated with Ad-EAT2 +Ad-Gag (358). Like TLRs, activation of the
SLAM receptor pathway in DCs and macrophages can also enhance the production of proinflammatory cytokines (373).
The biochemical mechanism and intracellular signaling pathway behind EAT-2’s ability
to function as a T cell (and possibly a B cell) stimulator in the face of CS protein over expression
is not fully elucidated, but is a question that has been unveiled by our studies. SLAM associated
proteins like EAT-2 are known to play a role in several novel immuno-modulatory pathways,
including the SLAM, CD22, and FcγRIIB (374-376). These pathways may not be subject to the
immune suppressive actions of CS protein possibly by virtue of its specific mode of action
relative to NF-κB and/or TLR activation pathways described earlier.
+
It has been established that greater numbers of CD8 T cells are required to police
infected hepatocytes and achieve long term protective immunity against malaria, emphasizing
+
the importance of inducing a large population of CD8 T cells capable of killing (377). There is
some evidence that improved protection is also related to increased breadth of the CMI response
in addition to the potency of the CMI response (309, 361, 378). Here, as an accessory to the
increased CMI response, we have demonstrated Ad-EAT2’s ability to stimulate increased T cell
responses against multiple CS protein epitopes. We not only observed an increase in the
+
percentage of CS protein specific CD8 T cells, but also improved in vivo CTL killing of CS
pulsed splenocytes from mice treated with Ad-CSP +Ad-EAT2. The use of EAT-2 to augment
+
CSP specific functional CD8 T cells may be of greatest importance in killing Plasmodium
infected hepatocytes, as these types of responses are not only positively correlated with
137
protective capability, but also may outweigh the need for induction of malaria antigen specific
antibody responses (337, 338, 377, 379).
Improvements over sole use of Ad-CSP to induce CS protein antigen specific B cell
responses were not achieved in mice treated with either Ad-CSP vaccine cocktail. However, covaccination with the Ad-CSP and Ad-EAT2 vectors at least prevented the loss of induction of CS
specific antibody responses noted after use of the Ad-GFP/rEA and Ad-CSP vaccine
combination. These results did not appear to be due to a skewing from Th1 to Th2 type antibody
response, as measured by IgG1/IgG2a ratios; there were also no observed differences in IFNγ
-
+
secreting CD8 CD3 T cells between treatment groups. Further research will need to be
performed to elucidate the reasons behind the observed antibody responses.
The importance of stimulating a strong cytotoxic T cell response against P. falciparum
infected hepatocytes is vital in creating a subunit based vaccine that is protective against malaria.
With this study we have successfully stimulated a CMI response to CS protein that can overcome
CS protein related adaptive immune response ablation and is even more potent than the previous
generation of rAd5s expressing CS protein. Incorporation of this new vaccine platform into
ongoing or future malaria vaccine trials could potentially achieve the levels of prophylaxis
needed to protect vulnerable populations against natural malaria infections. Future studies will
need to be performed to assess this platforms ability to protect larger animals challenged with
malaria.
138
Acknowledgements:
We wish to thank Michigan State University Laboratory Animal support facilities for their
assistance in the humane care and maintenance of the animals utilized in this work, the NIH Core
Tetramer Facility at Emory University for manufacturing the NYDNAGTNL tetramer, and the
Michigan State University flow cytometry facility for their assistance with the multiple
experiments. A.A. was supported by the National Institutes of Health grants RO1 AR056981 and
P01 CA078673, the MSU Foundation, as well the Osteopathic Heritage Foundation. YAA was
supported by the King Abdullah bin Abdulaziz Scholarship, Ministry of Higher Education,
Kingdom of Saudi Arabia.
139
Chapter IV
TRIF is a critical negative regulator of TLR agonist mediated activation of dendritic cells
in vivo.
This chapter is the edited version of a research article that was published in PLoS ONE Journal,
Volume 6, Issue 7 (e22064), July 8, 2011.
Authors: Aldhamen Y.A.*, Seregin S.S. *, Appledorn D.M., Aylsworth C.F., Godbehere S., Liu
C.J., Quiroga D., and Amalfitano A. *Authors contributed equally to the contents of this paper.
140
4.1.
Introduction
There is a great need to develop more efficient vaccines to combat or prevent infections
by a number of detrimental pathogens that continue to plague mankind (380-382). The use of
novel adjuvants capable of beneficially stimulating the immune system to maximize efficacy of
various vaccination strategies is a rapidly developing field. Most adjuvants augment the
induction of innate immune responses by triggering robust activation of dendritic cells (DCs) and
macrophages, actions that can result in improved induction of antigen specific adaptive immune
responses. Upon migration to the draining lymph nodes, these highly active antigen presenting
cells (APCs) are capable of presenting specific antigens to responsive T cells, thereby generating
significant pools of antigen-specific T cells (381, 383). Incorporation of Toll-like receptor (TLR)
ligands into vaccine formulations represent a class of adjuvants proposed for usage in next
generation vaccines. This is primarily due to TLRs being expressed at high levels on important
immune cell types (DCs, macrophages, NK cells) and their ability to potently activate the innate
immune system (383-385).
In light of these facts, the recombinant, Eimeria tenella derived antigen (rEA) has been
proven to be capable of inducing IL-12p70 production, enhancing Th1 cellular responses, and
yielding protection against Toxoplasma gondii infection in mice (134). rEA has also been shown
to be an efficient immunomodulator, having both antiviral and anti-cancer properties (131, 135,
136). Previous studies have also shown that HIV-Gag-specific T cell responses are significantly
increased when rEA formulations are administered together with the antigen (133, 139).
Moreover, rEA showed no evidence of toxicity in pre-clinical (386) and clinical trials (137).
Specifically, no severe adverse reactions were reported in human clinical trials despite detection
of increased IL-12 responses in 30% of the treated cancer patients (137).
141
The rEA protein has a relatively high amino acid sequence homology (67%) and shares
very similar biological activities in vitro and in vivo with T. gondii-derived profilin-like protein,
both of which trigger potent IL-12 responses in DCs. The profilin induced responses were
completely dependent upon the adaptor protein MyD88 and at least partially mediated via
TLR11 (138). Moreover, it has been shown in vitro that human TLRs (TLR2, TLR3, TLR4,
TLR5, TLR7, TLR8 and TLR9) do not transduce rEA signaling (135). Therefore, TLR11 has
been suggested as the rEA receptor mediating rEA signaling, but this notion remains to be
confirmed. Since no functional human TLR11 homolog has been discovered, these facts leave
unidentified the mechanism underlying rEA action in humans, and opens a discussion regarding
other pattern recognition receptors (PRRs) that may be involved in rEA signaling (135).
Additionally, it is not known what cell types are primarily responsible for mediating rEAtriggered responses in vivo.
MyD88 and TRIF are two adaptor proteins which primarily mediate the signaling derived
from activation of many pattern recognition receptors (PRRs), including TLRs (385). We have
investigated if rEA requires either of these two proteins to trigger immune responses in vivo, and
have found that all rEA-triggered immune responses are dependent on MyD88 functionality
(including the rapid activation of DCs, macrophages, NK, NKT, T and B cells, the induction of
pro-inflammatory
cytokine/chemokine
releases,
as
well
as
Erk1/2
phosphorylation).
Surprisingly, we discovered that functional TRIF protein acts to suppress rEA induction of these
same responses; thereby unveiling a novel inhibitory role for TRIF during rEA mediated
signaling. We also present evidence that TRIF may similarly suppress TLR activations by other
known TLR ligands. Together the findings highlight the complexities underlying adjuvant
activation of the innate immune system, as well suggests that simple notions of augmenting or
142
modifying adjuvant activity by use of TLR system agonists or antagonists may be complicated
by these complex molecular mechanisms.
143
4.2.
Results
The purpose of this study was to identify the impact of rEA stimulation on host immune
systems, to define important cell types that respond to or modulate rEA-driven activation, and to
identify signaling pathways responsible for activation of the immune system in response to rEA.
To investigate this, C57BL/6 mice were each intraperitoneally (IP) injected with 100 ng of
purified, rEA protein. Splenocytes were harvested at 6 hpi and flow cytometry was performed as
+
detailed in Materials and Methods. We identified that activation of murine DCs (CD11c ,
-
-
-
CD11b , CD19 , CD3 ) in response to rEA was completely dependent on the presence of full
+
MyD88 functionality. Specifically, we found significant increases in the percent of CD40 ,
+
+
CD80 and CD86 DCs (as well as induction of the expression of these molecules per cell as
measured by Mean Fluorescent Intensity [MFI]) in rEA treated wild type (WT) mice, but no such
increases were observed in rEA treated MyD88 knockout (KO) or MyD88/TRIF double
knockout (DKO) mice, each as compared to mock-injected animals. Surprisingly, we detected
significant (p<0.001) increases in DC activation after identical rEA treatments of TRIF-KO mice
(as compared to WT mice), as measured by CD40, CD80 and CD86 surface staining.
Furthermore, the amount of CD86 expression per cell was significantly (p<0.001) increased in
rEA treated TRIF-KO mice, as compared to rEA treated WT mice (Figures 31). rEA stimulation
also increased the percent of MHC-II presenting DCs in WT and TRIF-KO mice, increases that
were not seen in the rEA-treated MyD88-KO or the MyD88/TRIF-DKO mice. The DCs from
rEA-treated TRIF-KO mice also had significantly (p<0.05) higher MHC-II surface expression
levels as compared to rEA treated WT mice (Figure 31).
144
Figure 31: TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation
of dendritic cells in vivo. C57BL/6 WT (N=3-4), MyD88-KO (N=3), TRIF-KO (N=3-4), and
MyD88/TRIF-DKO (N=4) mice were injected with 100 ng of rEA. Splenocytes were harvested
at 6 hpi, processed, stained for expression of surface markers, and FACS sorted as described in
Materials and Methods. All genotype mock-injected mice (N=2-3) were included in analysis.
One of two representative experiments is shown. Separate sets of WT mice were utilized for
+
-
-
comparison with each knockout genotype. Activation of CD11c , CD19 , and CD3 DCs is
shown. The bars represent Mean ± SEM. Statistical analysis was completed using a two tailed
homoscedastic Student’s t-tests. *, ** - Indicate values statistically different from those in mock
injected animals (of the same genotype), p<0.05, p<0.001 respectively.
145
TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation of
dendritic cells in vivo.
146
+
-
-
rEA-mediated activation of splenic macrophages (CD11b , CD19 , CD3 ) was also
completely dependent on MyD88, as confirmed by lack of macrophage activation in response to
rEA stimulation in the MyD88-KO or the MyD88/TRIF-DKO mice. In contrast, WT mice
injected with rEA experienced a dramatic increase in the levels of CD80, CD86, and MHC-II, on
the surface of splenic macrophages (MFI), as well as in the percent of macrophages, expressing
the CD40, CD80, and CD86 activation markers. Similar to observations in DCs, macrophages
derived from rEA-treated TRIF-KO mice were activated to levels that were significantly higher
than levels measured in WT mice treated with rEA. Not only were the amounts of CD40expressing macrophages increased, but also both the percentages of CD80 and CD86 expressing
macrophages (p<0.01) and amount of these markers per cell (MFI, p<0.05) were significantly
increased in rEA treated TRIF-KO mice as compared to rEA-treated wild type mice, (p<0.05)
(Figures 32). These experiments unveiled an important, not previously described, role of TRIF as
a suppressor of rEA-induced TLR/MyD88 signaling in DCs and macrophages in vivo.
Figure 32: TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation
of macrophages in vivo. C57BL/6 WT (N=3-4), MyD88-KO (N=3), TRIF-KO (N=3-4), and
MyD88/TRIF-DKO (N=4) mice were injected with 100 ng of rEA. Splenocytes were harvested
at 6 hpi, processed, stained for expression of surface markers, and FACS sorted as described in
Materials and Methods. All genotype mock-injected mice (N=2-3) were included in analysis.
One of two representative experiments is shown. Separate sets of WT mice were utilized for
comparison with each knockout genotype. Activation of macrophages is shown. The bars
represent Mean ± SEM. Statistical analysis was completed using a two tailed homoscedastic
Student’s t-tests. *, ** - Indicate values significantly higher (#, ## - lower) from those in mock
injected animals (of the same genotype), p<0.05, p<0.001 respectively.
147
Figure 32: TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation
of macrophages in vivo.
148
Interestingly, we found that baseline levels of MHC-II expression on splenic DCs were
significantly increased in untreated, MyD88-KO and MyD88/TRIF-DKO mice (amount of
MHCII per cell, p<0.01, Figure 22) as compared to untreated WT mice or untreated TRIF-KO
mice (Figures 31,32 and 33). Additionally, the MyD88/TRIF-DKO mice also had higher baseline
levels of CD40 expression in DCs (Figure 33). This phenomenon was not unexpected, as we had
previously noted increased baseline levels of the MHCII -chain (three-fold higher) in MyD88KO mice, as confirmed by microarray transcriptome analysis and flow cytometry based analyses
((181) and data not shown). Potentially, these baseline changes may be due to lack of presence of
these adaptors during normal mouse development.
Figure 33: TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation
of dendritic cells in vivo (MFI). C57BL/6 WT (N=3-4), MyD88-KO (N=3), TRIF-KO (N=3-4),
and MyD88/TRIF-DKO (N=4) mice were injected with 100 ng of rEA. Splenocytes were
harvested at 6 hpi, processed, stained for expression of surface markers, and FACS sorted as
described in Materials and Methods. All genotype mock-injected mice (N=2-3) were included in
analysis. One of two representative experiments is shown. Separate sets of WT mice were
utilized for comparison with each knockout genotype. Mean Fluorescent Intensity (MFI) is
shown and is indicative of amount of analyte per cell. The bars represent Mean ± SEM.
Statistical analysis was completed using two-tailed homoscedastic Student’s t-tests. *, ** Indicate values statistically different from those in mock-injected animals (of the same
genotype), p<0.05, p<0.001 respectively.
149
Figure 33: TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation
of dendritic cells in vivo (MFI).
150
The unexpected result of TRIF being a negative regulator of rEA-triggered activation of
DCs and macrophages, prompted us to evaluate if TRIF reduces rEA-induced activation of other
important immune cells, including effector NK cells, as well as NKT, T and B cells. We have
previously shown that rEA protein activates splenic and hepatic NK and NKT cells within 6
hours post-injection in WT mice (133). In this study, rEA injections into MyD88-KO and
MyD88/TRIF-DKO mice yielded only baseline activation levels of CD69+ NK, NKT, T and B
cells. This contrasted with a significant activation of these same cell types in WT mice treated
with rEA protein (Figure 34). Similarly, the amount of IFN production from NK and NKT cells
was not increased in the MyD88-KO or the MyD88/TRIF-DKO mice in response to rEA,
whereas WT mice had dramatically increased numbers of IFN secreting NK (from 1% to 2030%) and NKT (from 1% to 2-5%) cells. In contrast, rEA treatment of TRIF-KO mice revealed
significant increases in the number of IFN producing NK cells (p<0.05), the amount of CD69
expression per NK cell (MFI, p<0.01), the number of cells (p<0.05) and amount (p<0.05) of
IFN-production from NKT cells, the number of cells (p<0.01) and amount (p<0.01) of CD69
expression from T cells, and the number of B cells expressing CD69 (p<0.01); all in comparison
to rEA-treated WT mice (Figure 34).
Figure 34: TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation
of NK, NKT, T, and B cells in vivo. C57BL/6 WT (N=3-4), MyD88-KO (N=3), TRIF-KO
(N=3-4), and MyD88/TRIF-DKO (N=4) mice were injected with 100 ng of rEA. Splenocytes
were harvested at 6 hpi, processed, stained for expression of surface markers (intracellular
staining was performed for IFN), and FACS sorted as described in Materials and Methods. All
genotype mock-injected mice (N=2-3) were included in analysis. Separate sets of WT mice were
151
utilized for comparison with each knockout genotype. Activation of NK, NKT, T, and B cells is
shown. The bars represent Mean ± SEM. Statistical analysis was completed using a two tailed
homoscedastic Student’s t-tests. *, ** - Indicate values statistically different from those in mock
injected animals (of the same genotype), p<0.05, p<0.001 respectively.
152
Figure 34: TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation
of NK, NKT, T, and B cells in vivo.
153
Within 6 hours of administration, rEA protein triggers significant production of proinflammatory cytokines and chemokines in mice. Specifically, circulating levels of IL12p70,
IL6, TNF, IFN, and IL2 were markedly elevated in rEA-treated mice (135, 386). Purified
murine DCs exposed to 0.2 ng/ml of rEA were shown to release significant amounts of IL12p70,
IL6, and IL2 (386). We confirmed and extended the observation that rEA triggers the release of a
wide spectrum of pro-inflammatory cytokines and chemokines, including IL6, IL12p40,
IL12p70, GCSF, IFN, IL2, IL1, IL1, IL10, IL13, GMCSF, KC, MCP1, MIP1, MIP1,
RANTES, and TNF. This effect was however, completely dependent on MyD88 given that all
of these analytes were induced in rEA-treated WT, but not in rEA-treated MyD88-KO or
MyD88/TRIF-DKO mice (Figure 35). Again, paralleling our previous results, the release of all
of these cytokines and chemokines was significantly increased in rEA-treated TRIF-KO mice,
and the majority of these analytes were elevated to levels that were significantly higher than
levels measured in rEA-treated WT mice. In particular, IL6 was induced to ~3 fold (p<0.001)
higher levels and IL12p40, GCSF, and IFN were induced to over 2 fold (p<0.001) higher levels
when comparing rEA-treated TRIF-KO mice to rEA-treated WT mice. Moreover, IL12p70, IL2,
IL1, IL1, and MIP1 were also produced at significantly higher levels in rEA-treated TRIFKO mice as compared to rEA treated WT mice (Figure 35).
Figure 35: TRIF negatively regulates rEA-mediated MyD88 dependent activation of proinflammatory cytokines and chemokines in vivo. C57BL/6 WT (N=9), MyD88-KO (N=3),
TRIF-KO (N=3-4), and MyD88/TRIF-DKO (N=4) mice were injected with 100 ng of rEA.
Plasma samples were collected at 6 hpi and were analyzed using a multiplexed bead array based
quantitative system. All genotype mock-injected mice (N=2-3) were included in analysis. One of
two representative experiments is shown. Statistical analysis was completed using a one-way
154
ANOVA with a Student-Newman-Keuls post-hoc test. The bars represent Mean ± SD. *, ** Indicate plasma cytokine values that are statistically different from those in mock injected
animals, p<0.05, p<0.001 respectively. No significant differences between mock-injected
animals of different genotypes were detected. No significant activation of cytokines was
observed in MyD88-KO and MyD88/TRIF-DKO animals.
155
Figure 35: TRIF negatively regulates rEA-mediated MyD88 dependent activation of proinflammatory cytokines and chemokines in vivo.
156
It is known that many of the rEA induced cytokines and chemokines are released by DCs
+
(Figure 35) (387). For that reason, we purified CD11c DCs from WT, MyD88-KO, TRIF-KO,
and MyD88/TRIF-DKO mice, and then stimulated the cells ex vivo with escalating amounts of
rEA protein. Utilizing an IL12p70 specific ELISA, we confirmed that in response to rEA, DC
production of this cytokine was completely dependent upon functional MyD88 (Figure 36A).
The minimal rEA dose in which WT mouse-derived DCs produced significant amounts of
IL12p70 was found to be 100 pg/ml, whereas TRIF-KO mouse-derived DCs only required a 10
pg/ml dose (10 fold less) for significant IL12p70 release. In an overall comparison to WT mouse
derived DCs, TRIF-KO mouse derived DCs had a significantly higher production of IL12p70 in
response to rEA. The most dramatic difference between these two groups was noted when DCs
were stimulated with a 0.2 ng/ml dose of rEA, which caused ~1300 pg/ml of IL12p70 to be
released from DCs derived from TRIF-KO mice as compared to ~600 pg/ml from DCs derived
from WT mice (p<0.01) (Figure 36A.). We have verified our ELISA-based data for IL12p70
independently, by Bioplex bead array. Again, we confirmed that MyD88-KO and the
MyD88/TRIF-DKO mouse derived DCs each respectively failed to produce significant levels of
pro-inflammatory cytokines and chemokines in response to rEA stimulation. Interestingly, we
also confirmed that these rEA mediated DC responses were also partially suppressed by TRIF,
as, IL2, IL6, IL12p40, IL12p70, IL1, IL1, and MIP1 were released to significantly (p<0.05)
higher levels in rEA-treated DCs derived from TRIF-KO mice, as compared to those derived
from WT mice (Figure 36B).
Figure 36: TRIF negatively regulates rEA-mediated MyD88 dependent activation of pro+
inflammatory cytokines and chemokines in dendritic cells. (A) CD11c dendritic cells were
isolated from C57BL/6 WT (N=2), MyD88-KO (N=2), TRIF-KO (N=2), and MyD88/TRIF157
DKO (N=2) mice, in vitro stimulated with rEA, then used to perform a IL12p70 ELISA as
described in Materials and Methods. One (of three) representative experiments is shown. The
bars represent Mean ± SD. Statistical analysis was completed using a two-way ANOVA with a
Bonferroni post-hoc test (genotypes x rEA treatments). *, ** - Indicate values that are
statistically different from those in unstimulated DCs (for the same genotype), p<0.05, p<0.001
respectively. #, ## - Indicate values statistically different from those in WT DCs (for the same
rEA dose), p<0.05, p<0.01 respectively. No significant differences between mock-injected
animals of different genotypes were detected. No significant activation of IL12p70 was observed
in MyD88-KO and MyD88/TRIF-DKO DCs. (B) DC culture media was collected at 18 hours
post-rEA stimulation (0.2 ng/ml) and was analyzed for cytokines/chemokines levels using a
multiplexed bead array based quantitative system. Statistical analysis was completed using a
one-way ANOVA with a Student-Newman-Keuls post-hoc test. The bars represent Mean ± SD.
*, ** - Indicate cytokine values that are statistically different from those in mock injected
animals, p<0.05, p<0.001 respectively. No significant differences between mock-injected
animals of different genotypes were detected. No significant activation of pro-inflammatory
cytokines was observed in MyD88-KO and MyD88/TRIF-DKO animals (only anti-inflammatory
IL10 cytokine was induced in MyD88-KO mice).
158
Figure 36: TRIF negatively regulates rEA-mediated MyD88 dependent activation of
pro-inflammatory cytokines and chemokines in dendritic cells.
159
To more fully investigate if TRIF suppressive effects were rEA-specific or a more global
phenomenon, we have specifically stimulated CD11c+ DCs, isolated from WT, TRIF-KO,
MyD88-KO or MyD88/TRIF-DKO mice with various common TLR4, TLR7/8, and TLR9
agonists. Specifically, LPS, R848 and ODN2006 were utilized in these experiments. All of these
TLR agonists were able to induce pro-inflammatory cytokine production after administration to
DCs, as compared to unstimulated DCs, all derived from WT mice. Importantly, however, DCs
derived from TRIF-KO mice had dramatically higher levels of secretion of pleiotropic proinflammatory cytokines, when stimulated with these same TLR agonists (Figure 37AB).
Specifically, ODN2006 stimulation resulted in significantly higher IL6, IL12p40, IL12p70 and
MIP1 levels; LPS stimulation significantly increased IL1, IL3, IL6, IL12p70 levels; R848
treatment resulted in significantly higher production of IL1, IL1, IL6, and MIP1; in DCs,
derived from TRIF-KO mice as compared to DCs derived from WT mice, as tested both by
ELISA (Figure 37A) or Bioplex analysis (Figure 37B and data not shown). DCs derived from
MyD88-KO or MyD88/TRIF-DKO mice did not show any significant cytokine activations when
stimulated with any of these TLR agonists (data not shown). Interestingly, lack of the TRIF
adaptor protein resulted in up to 2 fold increases in cytokine production from DCs, when
stimulated with LPS or ODN2006 (i.e. IL12p70), indicating that the suppressive role of TRIF in
this cell type might be immunologically significant.
Figure 37: TRIF negatively regulates cytokine production by DCs, triggered by several
+
common TLR agonists. (A) CD11c dendritic cells were isolated from C57BL/6 WT (N=3),
MyD88-KO (N=3), TRIF-KO (N=3), and MyD88/TRIF-DKO (N=3) mice, in vitro stimulated
with various TLR agonists, and used to perform a IL12p70 ELISA as described in Materials and
Methods. The bars represent Mean ± SEM. Statistical analysis was completed using a one-way
160
ANOVA with Student-Newman-Keuls post-hoc test. ** - Indicate values that are statistically
different from those in unstimulated DCs (for the same genotype), p<0.001. No significant
differences between mock-injected animals of different genotypes were detected. No significant
activation of IL12p70 was observed in MyD88-KO and MyD88/TRIF-DKO DCs. (B) DC
culture media was collected at 15 hours post stimulation with various TLR agonists (rEA, LPS,
ODN2006) and was analyzed for cytokines/chemokines levels using a multiplexed bead array
based quantitative system. Statistical analysis was completed using a one-way ANOVA with a
Student-Newman-Keuls post-hoc test. The bars represent Mean ± SD. *, ** - Indicate cytokine
values that are statistically different from those in mock injected animals, p<0.05, p<0.001
respectively.
161
Figure 37: TRIF negatively regulates cytokine production by DCs, triggered by several
common TLR agonists.
162
Extensive past research has demonstrated that the major signaling pathways activated
downstream of TLR adaptor proteins (e.g. MyD88, TRIF) are the NFB and MAPK pathways
(8). Along with many other biological roles, these pathways promote production of cytokines
and chemokines as well as proliferation, maturation, and development of various immune cells.
To identify which of the several signaling pathways may be activated in response to rEA in vivo,
we IP injected WT or MyD88-KO mice with 100 ng of rEA, and then collected spleen and liver
tissues at various time points (0-120 minutes) post-injection. We found that rEA-triggered
Erk1/2 phosphorylation was MyD88 dependent, as confirmed by increased Erk1/2
phosphorylation in rEA-treated WT mice, but not in the MyD88-KO mice (Figure 38).
Figure 38: rEA-triggered Erk1/2 phosphorylation is MyD88 dependent. C57BL/6 WT or
MyD88-KO mice were injected with 100 ng of rEA. Spleen (A) and liver (B) tissues were
collected at the indicated time points and processed as described in Materials and Methods. pErk1/2 and Erk2 levels were determined by Western blot analysis using LI-COR Odyssey. To
control for loading, quantification was performed after normalizing the pErk1/2 to Erk2 levels.
Three independent experiments representative of this data are shown. (C) Representative blots:
spleen (top), liver (bottom).
163
Figure 38: rEA-triggered Erk1/2 phosphorylation is MyD88 dependent. C57BL/6 WT or
MyD88-KO mice were injected with 100 ng of rEA.
164
4.3. Discussion
The ideal adjuvant (from Latin “adjuvare”, meaning “to enhance”) is an agent that is
capable of dramatically enhancing both cellular and humoral adaptive immune responses to coadministered antigens, thereby providing more efficient and long-term protection against specific
pathogens. Aluminum salts, discovered to be potent adjuvants in 1920s, remained the only FDA
approved adjuvant for many decades and still represent one of the few in clinical use. A barrier
in adjuvant research has been that the mechanism of action of many adjuvants remained poorly
understood. Earlier studies showed that alum and squalene-based emulsion MF59 promote
recruitment and increase antigen uptake by APCs, induce cytokine and chemokine secretions,
and the expression of adhesion molecules involved in migration of leukocytes (388). More
recently it has also been confirmed that alum and squalene based adjuvants may use the NODlike receptor protein 3 (NLRP3) inflammasome pathway to activate the innate immune system
(389, 390).
The Eimeria tenella derived protein, was isolated from bovine small intestinal extracts
and was shown to have remarkable anti-cancer activity and be a potent stimulator of innate
immune responses in various mouse models in vitro and in vivo (386, 391, 392). The rEA
protein, we believe, might possess all the properties of an ideal immunologic adjuvant, as it can
be fairly inexpensive to produce, is extremely stable and can be stored for long periods of time
(over 24 month, data not shown) without losing activity. The rEA has been shown to be safe and
very well-tolerated in human clinical trials (137). In mice, rEA augments activation of the innate
immune system, presumably by activating PRRs (TLRs and/or possibly others) and thereby
increasing adaptive immune responses to co-administered antigens (133, 139). It has been
suggested that TLR11 is involved in rEA signaling (135), a notion that is primarily based on high
165
sequence homology (67%) between rEA and T. gondii profilin-like protein, the latter being the
only confirmed ligand for TLR11 (138).
In this study we confirmed that administration into mice, or direct exposure of immune
cells to rEA protein results in (1) activation of important immune cell types (DCs, macrophages,
NK, NKT, B and T cells), including (2) pro-inflammatory cytokines/chemokines release, both
globally and specifically by DCs, and highly robust IFN production by NK cells, and (3) Erk1/2
phosphorylation. Therefore, we showed that rEA, similarly to other TLR-agonist-based
adjuvants, activated an innate immune profile that resulted in robust activation of innate immune
cells and induced multiple cytokines/chemokine pathways. Our results also confirmed that these
responses are completely dependent upon MyD88, as genetic knockout of this adaptor protein
results in complete ablation of these responses both in vitro and in vivo.
When we similarly investigated the role of TRIF, the other major TLR adaptor protein,
we encountered unexpected results. Specifically, TRIF-KO mice showed dramatic increases, in
immune cell activation and other rEA triggered responses, when compared to rEA treated WT
mice. Specifically, we found that IL6, IL12p40, IL2, IL1, IL1, and MIP1 production was
induced by rEA treatment in TRIF-KO mice in vivo and in DCs derived from these mice in vitro
to significantly higher levels as compared to similar assays performed in rEA-treated WT mice.
Note, that rEA mediated induction of IL12p70, a cytokine abundantly produced by DCs that
activates NK cells, was also found to be negatively regulated by TRIF after exposure to rEA
(387, 393).
Therefore, in response to rEA-mediated stimulation, TRIF acts as a suppressor of the
rEA-induced (MyD88-dependent) activation of DCs, macrophages, NK, NKT, T, and B cells in
vivo. Despite the lack of TRIF activity in MyD88/TRIF-DKO mice, rEA responses were still
166
ablated which indicates that in the absence of MyD88, TRIF cannot carry out its suppressive
activity. Since the lack of TRIF protein does not rescue the phenotype in MyD88/TRIF-DKO
mice, this confirms an essential role of MyD88 in mediating these responses and suggests that
TRIF protein acts as a suppressor downstream of MyD88 and/or MyD88 and TRIF adaptor
molecules orchestrating the induction of pro-inflammatory immune responses following rEA
injection.
Numerous studies have described important roles for TRIF as a TLR system adaptor
protein that acts to enhance TLR-based signaling (predominantly when TLR3 and TLR4 ligands
are interrogated, thereby promoting antimicrobial responses (385, 394, 395). Specifically, TRIFKO mice showed dramatically impaired lung clearance of Pseudomonas aeruginosa infections; a
response that is correlated with blunted cytokine induction (e.g. RANTES, IL1, MIP2) and
reduced NFB activation present in both alveolar and peritoneal macrophages from these mice
(396). Lack of TRIF protein also results in reduced induction of antigen specific humoral and
cellular immune responses in other models (397). Specifically, T cells derived from TRIF-KO
mice had dramatically reduced IFN production and CXCR3 expression upon antigen/LPS
treatment as compared to WT mice (398). In DCs, TRIF functionality was confirmed to be
important for upregulation of CD40 and CD86 co-stimulatory molecules (399). Moreover, TRIF
protein along with IPS1 (RIGI/Mda5 pathway protein) are key adaptors in mediating Poly-ICtriggered adjuvant effects (397).
Conversely, there is very little data available that describes suppressive roles of TRIF
protein on TLR/PRR signaling. Specifically, we have previously demonstrated that a lack of
functional TRIF protein results in increased transgene (e.g. -Gal) specific IgG titers in mice
injected with Ad5-LacZ, suggesting that TRIF may act as a negative regulator of Ad-mediated
167
antibody responses in mice (400). Other researchers have demonstrated that TRIF has an
inhibitory role in TLR5-mediated responses through induction of TLR5 degradation (401). This
phenomenon may not be limited to TLR5, as it has been suggested that TRIF can also induce
degradation of other TLRs, including TLRs 3, 6, 7, 8, 9, and 10 (401). To determine if the
suppressive role of TRIF in DCs is more global than previously considered, we have stimulated
isolated DCs with TLR4 (LPS), TLR7/8 (R848) or TLR9 (ODN2006) agonists and found that
the presence of the TRIF adaptor protein significantly suppresses release of pleiotropic proinflammatory cytokines by DCs in response to these agonists. These studies suggest that
suppressive activities of TRIF, in regard to pathogen-induced innate immune responses, may be
more prevalent than currently appreciated.
Activation of pro-inflammatory cytokines/chemokines is a critical step during DC
activation/maturation. It has been suggested that TRIF may be required for inducing
immunological tolerance by augmenting IL10 production (402, 403). We found, however, that in
rEA (or other TLR agonist) treated TRIF-KO mice, amounts of IL10 (both in plasma and in
isolated DCs) were indistinguishable to identically (rEA) treated WT mice.
To successfully bridge DC activation to induction of substantial adaptive immune
responses (e.g. T cell activation), it is essential to have induction of co-stimulatory molecules
such as CD40, CD80, and CD86 on the surface of DCs. In this study, we have shown that
administration of rEA results in robust maturation of DCs, as evidenced by not only increased
expression of co-stimulatory molecules on these DCs, but also that rEA increased cytokines and
chemokine production. While these responses were completely abrogated in rEA-treated
MyD88-KO mice, most of them were dramatically enhanced in TRIF-KO mice when compared
to rEA treated WT mice.
168
From this information, we are proposing a model of rEA signaling in DCs. The rEA
protein likely interacts with a PRR, (likely TLR11 in mice, unknown in humans (135)), that
senses rEA. The MyD88 adaptor protein gets recruited which allows MAP kinases downstream
of MyD88 to become activated (pErk1/2, p38). pErk1/2 is capable of activating various
transcription factors (e.g. AP1) which further activates pro-inflammatory genes, including those
of pro-inflammatory cytokines/chemokines (395, 398, 404, 405). In contrast, functional TRIF
protein acts as a negative regulator of the rEA-induced signaling. As a result of TRIF’s inhibitory
effects, DCs have a reduction in surface expression of maturation markers, as well mitigated
release of pro-inflammatory cytokines/chemokines (387) (Figure 39).
Figure 39: TRIF acts as a negative regulator of rEA-induced signaling and downstream
responses in DCs: model of action. rEA is a protein derived from Eimeria tenella (133, 386)
and is highly homologous to Toxoplasma gondii profilin-like protein (138). T. gondii has been
shown to signal, at least in part, through TLR11; therefore, it is likely that TLR11 is one of the
main pattern recognition receptors (PRRs) utilized by rEA. We have shown that rEA-triggered
responses in vivo are completely dependent on MyD88. MAP kinases consequently become
activated downstream of MyD88. Importantly, functional TRIF protein inhibited rEA-mediated
signaling. DCs are a major cell type in mediating rEA responses and, under TRIF’s inhibitory
effects, have mitigated induction of surface expression of maturation markers and stunted release
of pro-inflammatory cytokines/chemokines. Release of these molecules is critical for rapid
amplification of immune responses and is mediated by autocrine and paracrine signaling (387).
We
have
confirmed
that
TRIF
protein
reduces
release
of
pro-inflammatory
cytokines/chemokines in response to rEA, resulting in reduced activation of NK, NKT, T, and B
cells as well as reduced IFN production by NK cells.
169
Figure 39: TRIF acts as a negative regulator of rEA-induced signaling and downstream
responses in DCs: model of action.
170
In conclusion, our murine models have shown that rEA activates multiple immune cell
types, stimulates pro-inflammatory cytokine/chemokine release, and triggers the MAPK pathway
in a MyD88 dependent manner. We have also confirmed that DCs are the main subset of innate
immune cells that mediate rEA-triggered responses, thus justifying future studies on isolated
DCs and the potential use of rEA adjuvant in a DC-vaccine setting (406). Importantly, our
studies of rEA unveiled a suppressive activity to the TRIF adaptor protein. This clearly justifies
future testing of specific TRIF inhibitors or knockdown models prior to rEA/antigen
administration as a means to further enhance induction of antigen specific adaptive immune
responses. Whether TRIF acts to negatively regulate other adjuvants or TLR-mediated
activations is a question that will require future investigations. The latter, however, highlights the
complexities of TLR adaptor functions, and should temper efforts that target these proteins with
agonists or antagonists, as the end result of such interventions may run counter to hoped for
outcomes, and could be detrimental in some situations (407)
171
Acknowledgements
We wish to thank the Michigan State University Laboratory Animal support facility for
their assistance in the humane care and maintenance of the animals utilized in this work. A. A.
was supported by the MSU Foundation, as well the Osteopathic Heritage Foundation. YAA was
supported by the King Abdullah bin Abdulaziz Scholarship, Ministry of Higher Education,
Kingdom of Saudi Arabia.
172
Chapter V
Preventing CRACC receptor upregulation in antigen presenting cells improves induction
of antigen specific adaptive immune responses by vaccines
.
173
5.1.
Introduction
The innate immune system relies heavily on a variety of transmembrane, intracellular, or
secreted pattern-recognition receptors (PRRs), each of which are vital for recognition of specific
molecular structures found on, or within potentially infectious agents, such as viruses or
microbes (408). Activation of these PRRs triggers signaling pathways that regulate the
transcription of pro-inflammatory cytokine and chemokines genes, as well as other innate
immune defense responses, responses that also help shape subsequent, antigen specific adaptive
immune responses, (5, 408). The innate immune system also has a key role in initiating and
orchestrating the adaptive immune responses to antigens presented during vaccinations (108,
385). These facts suggest that specific modulation of innate immunity during vaccination may
allow for the development of improved therapeutic and preventative vaccines (120). A highly
characterized family of innate immune receptors, the Toll-like receptors (TLRs) , have been
targeted for modulation in pre-clinical and clinical vaccine applications (4). Triggering TLR
signaling by use of specific agonists or over-expressing TLR adaptors, (MyD88 or TRIF), has
been shown to induce pro-inflammatory cytokine secretion and to augment the adaptive immune
responses toward target antigens (133, 293, 409).
Another important family of immunoreceptor that plays a critical role in immune
regulation is the signaling lymphocytic activation molecule (SLAM) family of receptors (289).
SLAM receptors function as adhesion molecules on the surface of many hematopoietic cells.
They serve as costimulatory molecules that regulate intracellular signaling pathways that govern
the function of T-, B-, NK-, macrophages, and dendritic cells (DCs) (410). The SLAM family of
receptors currently comprise six distinct innate and adaptive immune-cell specific members,
respectively named SLAM (CD150), 2B4 (CD244), Ly9, CD84, NTB-A (natural killer, T and B
174
cell antigen; Ly108 in the mouse) and CRACC (CD2-like receptor activating cytotoxic cells)
(149, 287). All SLAM members except 2B4, (which interacts with CD48) are self-ligands. They
initiate intra-cellular signaling via recruitment of specific adaptors (287). For example, activation
of the CRACC receptor by CRACC-specific antibodies or self ligation to CRACC being
expressed on a neighboring cell promotes NK cells cytotoxicity (150, 151, 411, 412). In addition,
in CD40L-activated human DCs, antibody-mediated ligation of SLAM (CD150) augmented the
secretion of pro-inflammatory cytokines IL-12 and IL-8 (291). Furthermore, the SLAM (CD150)
receptor was also found to regulate the production of IL-6 and IL-12 by mouse peritoneal
macrophages (292).
The SLAM-associated protein family of adaptors includes three members named SAP,
Ewing’s sarcoma-associated transcript-2 (EAT-2), and EAT-2-related transducer (ERT; ERT is
however a non-functional pseudo-gene in humans). These adaptors associate with
phosphorylated tyrosine-based motifs (‘immunoreceptor tyrosine based switch motifs’ (ITSMs))
in the cytoplasmic domains of SLAM family receptors with high affinity and specificity. All
SLAM receptors can interact with either of the adaptors, except CRACC which interacts only
with EAT-2 and not SAP (140, 150, 151). SLAM family of adaptors regulate SLAM induced
intracellular signaling in a variety of immune cells (286). EAT-2 and ERT directly transduce
SLAM initiated signals via phosphorylation of tyrosine residues located in their short carboxylterminal tails (165). In contrast, SAP regulates SLAM signaling by recruiting the protein tyrosine
kinase FynT (413).
Since EAT-2 is the only known SLAM-associated adaptor protein expressed in DCs and
macrophages, it has been proposed that EAT-2 facilitates SLAM dependent pro-inflammatory
cytokine expression in these cell types (290). In addition, SLAM receptor (CD150) has been
175
found to function as a critical microbial sensor that positively regulates bacterial killing by
macrophages (414). In a recent report, it was also shown that the SLAM family member CRACC
positively regulates NK cell function by a mechanism dependent on the adaptor EAT-2, but not
the related adaptor SAP (151). Furthermore, the CRACC receptor can also have inhibitory
functions in T cells during antigen presentation. Interestingly, T cells do not express the EAT-2
adaptor (151).
We have previously described a novel immunostimulatory function for the SLAM family
receptors adaptor EAT-2 in a vaccine model system in mice, a system in which over-expression
of EAT-2 resulted in improved induction of robust antigen specific adaptive immune responses
after Adenovirus (Ad) mediated transfer and expression of antigen encoding genes (415). In this
study, we set out to investigate the mechanism of action of EAT-2. Specifically, we studied the
impact of EAT-2 over-expression in DCs and macrophages function, to determine how EAT-2
functions to alter induction of antigen specific adaptive immune responses. The results of our
experiments show that Ad mediated expression of EAT-2 activate signaling cascades that induce
downstream activation of ERK dependent signaling pathways in DCs and macrophages. This
activity correlated with suppressed expression of the CRACC receptor on DCs and macrophages,
a response that is completely dependent upon the interaction between the EAT-2 adaptor SH-2
domain and the phosphorylated ITSMs of the SLAM receptors. Thus, EAT-2 functions are not
only required to activate NK cell effector and regulatory functions, but, like other PRR adaptors,
induces critical regulatory pathways crucial for the improved function of DCs and macrophages
during antigen presentation to the adaptive immune system.
176
5.2.
Results
SLAM family receptors are self ligands (416) and homophilic interactions between these
receptors triggers immune cell activation (417). Most SLAM family receptors are expressed on
dendritic cells and macrophages (410). Therefore, we set out to evaluate the mechanisms
underlying EAT-2 involvement in the process of antigen presentation in APCs. We initially
investigated the impact that EAT-2 over-expression has on the expression levels of SLAM
receptors in macrophages. To define the expression pattern of these receptors, RAW264.7
macrophages were mock infected, or infected with EAT-2 expressing Ads, or a control Ad vector
(Ad-Null). Six hours post infection (hpi), the expression levels of the SLAM family receptors
SLAM, 2B4, CRACC, CD84, and Ly-9 were analyzed by quantitative RT-PCR as previously
described (101). We found that Ad vector infection itself significantly (p<0.001) induced the
expression of the SLAM family member CRACC, but not other SLAM family members in
macrophages (Figure 40A). In dramatic contrast, infection of macrophages with an EAT-2
expressing Ad significantly reduced Ad induction of CRACC receptor gene expression
(p<0.001) (Figure 40A). We also attempted to evaluate the expression level of the SLAM family
member Ly108, however its expression was not detectable in these cells, a result consistent with
previously published studies (418). A time course study confirmed and extended these results.
For example, at 3 hpi the expression level of the CRACC receptor was significantly and
equivalently induced (p<0.01) by both the Ad-EAT2 and Ad-Null vectors (Figure 40B and
supplementary Figure 40A). However, at later time points (6, 15, and 24 hpi), Ad infection
continued to significantly (p<0.001) induce CRACC receptor expression, while Ad infection
coupled with EAT2 overexpression significantly (p<0.001) reduced CRACC expression at these
same time points (Figure 40B).
177
Figure 40: EAT-2 functions as a negative regulator of Adenovirus mediated induction of
CRACC receptor expression on macrophages in vitro. RAW264.7 cells (300,000 cells/ well)
were mock infected or infected with 20,000 vector particles/ cell of Ad-EAT2 (black) AdEAT2(R31Q) (dash line) or Ad-Null (gray). (A) Quantitative RT-PCR for SLAM family
receptors six hours post infection (6 hpi). (B) Time course study for CRACC receptor expression
in RAW264.7 macrophages. Statistical analysis was completed using One Way ANOVA with a
student- Newman-Keuls post-hoc test, p<0. 05 was deemed a statistically significant difference.
Data are representative of five independent experiments with similar results. Samples were
plated in quadruplicate and are expressed as mean ± SD (* denotes p<0. 05, ** denotes P< 0.001
statistically different from mock infected cells).
178
Figure 40: EAT-2 functions as a negative regulator of Adenovirus mediated induction of
CRACC receptor expression on macrophages in vitro.
179
By 15 hpi, both Ad-EAT2 and Ad-Null induced similar levels of ADAR, ICAM-1, NOD1, TNF-α, IL-6, and IL-15 genes, results that support the notion that EAT-2 over-expression
specifically repressed only CRACC receptor RNA levels after Ad infection of macrophages
(Figure 41).
Figure 41: EAT-2 over-expression induces similar transcript levels of innate immune
responses genes as compared to adenovirus control. RAW264.7 cells (300,000 cells/ well)
were mock infected or infected with 20,000 vector particles/ cell of Ad-EAT2 (black) or Ad-Null
(gray). Quantitative RT-PCR for ADAR, ICAM, and NOD-1, TNF-a, IL-6 and IL-15 gene
expression in RAW264.7 macrophages at 15 hpi following Ads infection. Statistical analysis was
completed using One Way ANOVA with a student- Newman-Keuls post-hoc test, p<0.05 was
deemed a statistically significant difference. Data are representative of two independent
experiments with similar results. Samples were plated in quadruplicate and are expressed as
mean ± SD (* denotes p<0.01, *** denotes P< 0.001 statistically different from mock infected
cells).
180
Figure 41: EAT-2 over-expression induces similar transcript levels of innate immune
responses genes as compared to adenovirus control.
181
We also directly measured EAT-2 transcript levels at the time points tested. We
observed minimal induction of EAT-2 (two fold over mock) following Ad-Null infection (Figure
42A), suggesting minor activation of EAT-2 signaling pathway following Ad infection. In
contrast, significant increases in EAT-2 transcript were detected in Ad-EAT2 transduced cells,
due to high level EAT-2 gene expression from the Ad vector (Figure 42B).
Figure 42: EAT-2 transcript levels following Ad-EAT2 and Ad-EAT2 (R31Q) infection
(A) EAT-2 transcript level at 6 and 15 hpi after Ad-Null infection. (B) Time course study for
EAT-2 transcript level in RAW264.7 cells after Ad-EAT-2 infection. (C) EAT-2 transcript level
at 15 hpi following Ad-EAT2 or Ad-EAT2 (R31Q) infection. Samples were plated in
quadruplicate and are expressed as mean ± SD.
182
Figure 42: EAT-2 transcript levels following Ad-EAT2 and Ad-EAT2 (R31Q) infection.
183
To evaluate if protein levels of the CRACC receptor correlated with CRACC receptor
RNA transcript levels, we utilized FACS analysis. RAW264.7 cells were infected with Ad-EAT2 or Ad-Null for 20 or 72 hpi. At 20 hpi, both Ad-EAT2 and Ad-Null significantly induced
(p<0.001) the cell surface expression of CRACC receptor to similar levels, (data not shown). At
72 hpi, Adenovirus infection significantly (p<0.001) induced the cell surface expression of
CRACC receptor (Figure 43A). Importantly, infection with Ad-EAT2 significantly (p<0.01)
reduced the number of CRACC expressing RAW264.7 cells as well as the number of CRACC
molecules per cell, as compared to Ad-Null infected cells (Figure 43A). In contrast, the cell
surface expression of 2B4, CD84, and Ly-9 were similarly expressed in both Ad-EAT2 and AdNull infected cells (data not shown). We also utilized a more relevant in vitro primary
macrophage cell culture system. Bone marrow cells harvested from C57BL/6 mice were
differentiated into macrophages (BMDMs). BMDMs were infected with Ad-EAT-2 or Ad-Null
for 72 hpi and the cell surface expression of CRACC receptor was evaluated by flow cytometry.
Similar to RAW264.7 cells, Ad-Null infection resulted in significantly (p<0.001) increased
expression of the CRACC receptor, while infection of BMDMS with Ad EAT-2 significantly
(p<0.001) reduced the number of CRACC expressing cells we detected, as well as the number of
CRACC molecules per cell detected by this method (Figure 43B). To confirm this mechanism is
+
not limited to one APC type, we isolated murine splenic CD11c DCs and evaluated CRACC
receptor cell surface expression by flow cytometry. Similar to macrophages, Ad infection of
+
CD11c DCs significantly (p<0.001) increased the cell surface expression of the CRACC
receptor (Figure 43C). Importantly Ad mediated transduction of EAT-2 significantly (p<0.05)
reduced CRACC receptor expression in these same cells (Figure 43C). Thus, EAT2 over-
184
expression by Ads prevents Ad induced transcription of the CRACC receptor gene, resulting in
reduced CRACC protein levels in Ad transduced macrophages and DCs in vitro.
Figure 43: EAT-2 over-expression reduce protein level of CRACC receptor on DCs and
macrophages in vitro. In vitro cultured (A) RAW264.7 macrophages, (B) murine bone marrow
+
derived macrophages (BMDMs), or (C) isolated murine splenic CD11c DCs, (300,000 cells/
well) were mock infected or infected with the Ad-EAT2 (black) or Ad-Null control (gray) for 72
hours at the multiplicity of infection (MOI) of 20,000 vector particles/ cell for RAW264.7 and
+
BMDMs or MOI of 5000 for CD11c DCs. At 72 hpi, cells were stained with APC-conjugated
CRACC specific antibody and analyzed on an LSR-II flow cytometer. (A) Expression of
CRACC receptor on RAW264.7 following infection with Ad-EAT2 or Ad-Null. (B) Expression
+
of CRACC receptor on BMDMs. (C) Expression of CRACC receptor on murine splenic CD11c
cells. Statistical analysis was completed using One Way ANOVA with a student- NewmanKeuls post-hoc test, p<0.05 was deemed a statistically significant difference. Data are
representative of three independent experiments with similar results. Samples were plated in
quadruplicate and are expressed as mean ± SD (*, denotes p<0.05, ** denotes P<0.01, ***
denotes P< 0.001 statistically different from mock infected cells).
185
Figure 43: EAT-2 over-expression reduce protein level of CRACC receptor on DCs and
macrophages in vitro.
186
To investigate whether EAT-2 over-expression negatively regulates CRACC receptor
expression in vivo, C57BL/6 mice were either mock injected (PBS), or intravenously injected
10
with 7.5 × 10
vps of Ad-EAT-2 or Ad-Null as previously described (103). At 10 hpi, Ad
infection itself significantly (p<0.01) increased the expression level of CRACC receptor on
+
-
CD11c CD11b DCs, as no significant differences were observed between Ad-EAT2 and AdNull infected mice at this time point (Figure 44A). Importantly, at 48 hpi, consistent with the in
+
-
vitro results, the level of CRACC receptor expression on CD11c CD11b DCs was significantly
reduced in Ad-EAT2 injected mice as compared to Ad-Null injected controls (Figure 44B).
These results further confirm that administration of Ad vectors induces CRACC expression, but
that EAT-2 over-expression prevents this induction, both in vitro and in vivo.
Figure 44: EAT-2 over-expression negatively regulates CRACC expression in dendritic
cells in vivo. C57BL/6 mice (n=4) were either mock injected, or intravenously injected with
10
7.5×10
vps of either Ad-EAT2 or Ad-Null vectors. Splenocytes were harvested at 10 hpi (A)
+
-
or 48 hours (B) post-injection and CRACC receptor expressing CD11c CD11b DCs was
identified by using a LSR-II flow cytometer. The bars represent mean ± SD. Statistical analysis
was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05
was deemed a statistically significant difference. * denotes p<0. 05, ** denotes p<0.01
statistically different from mock injected animals.
187
Figure 44: EAT-2 over-expression negatively regulates CRACC expression in
dendritic cells in vivo.
188
The tyrosine-based motif (ITSM) present in the cytoplasmic domain of SLAM family
receptors interacts with the Src homology 2 (SH2) domains present in SAP adaptors (140, 152).
To investigate whether this specific interaction was required for EAT-2 to prevent Ad induced
expression of the CRACC receptor on APCs, we generated a mutant form of the EAT-2 adaptor,
EAT-2(R31Q) that contains a missense mutation that replaces the positively charged arginine
residue present at this location with a glutamine. Based upon studies in the highly homologous
SAP adaptor, we hypothesized that the mutation would disrupt the putative phosphotyrosinebinding pocket of EAT-2 (419). Once generated by targeted mutagenesis, the Ad vector
expressing EAT-2(R31Q), Ad-EAT2 (R31Q), was generated and successfully purified to high
titer. We first confirmed that the Ad-EAT2 (R31Q) mutant virus expressed similar levels of the
EAT-2 transcript per vector particle number as the Ad vector expressing the wild type version of
EAT-2 (Figure 45A). We then evaluated CRACC receptor expression on RAW264.7 cells at 15
hpi following identical infection with Ad-EAT2, Ad-EAT2 (R31Q), or Ad-Null control.
Consistent with the results obtained previously (see Figure 41A and B), infection with wild type
(WT) EAT2 expressing Ads significantly (p<0.01) prevented Ad-mediated induction of CRACC
receptor compared to Ad-Null infected cells (Figure 45B). Conversely, the inhibitory function of
EAT-2 was completely eliminated when the identical experiment was performed utilizing the Ad
expressing the EAT-2 phosphotyrosine binding pocket mutant (Figure 45B). Importantly, no
significant differences in CRACC levels were observed between Ad-Null and Ad-EAT2 (R31Q)
viruses (Figure 45B). We also evaluated the protein levels of CRACC at 72 hpi by flow
cytometry. Consistent with the transcript level, infection with Ad-EAT2 (R31Q) virus resulted
in enhanced protein levels of CRACC (p<0.001) as compared to Ads expressing the WT version
of EAT-2 (Figure 45C). These results strongly suggest that CRACC receptor down-regulation by
189
EAT-2 is specifically regulated by direct interaction between the SH-2 domain of EAT-2 and the
phosphorylated ITSMs of SLAM receptors present on APCs.
Figure 45: Mutant form of EAT-2 adaptor does not prevent CRACC upregulation by Ads.
RAW264.7 cells (300,000 cells/ well) were mock infected or infected with 20,000 vector
particles/ cell of Ad-EAT2 (black) Ad-EAT2(R31Q) (dash line) or Ad-Null (gray). (A) EAT-2
transcript level at 15 hpi following Ad-EAT2 or Ad-EAT2 (R31Q) infection. (B) Quantitative
RT-PCR for CRACC transcript at 15 hpi derived from Ad-EAT2, Ad-Null, or Ad-EAT2 (R31Q)
infected RAW264.7 cells. (C) FACS analysis of CRACC receptor on RAW264.7 following
infection with Ad-EAT2, Ad-EAT2 (R31Q) or Ad-Null. Statistical analysis was completed using
One Way ANOVA with a student- Newman-Keuls post-hoc test, p<0.05 was deemed a
statistically significant difference. Data are representative of three independent experiments with
similar results. Samples were plated in quadruplicate and are expressed as mean ± SD (*,
denotes p<0.05, ** denotes P<0.01, *** denotes P< 0.001 statistically different from mock
infected cells).
190
Figure 45: Mutant form of EAT-2 adaptor does not prevent CRACC upregulation by Ads.
191
The signaling pathways that are activated by EAT-2 in APCs are not completely defined.
Previous reports have shown that downstream of SLAM family receptors, EAT-2 activates the
Src-kinase FynT (420), phospholipase C gamma (150, 167), and PI3K (150) signaling pathways
in NK cells. We set out to investigate which signaling pathway(s) are activated by EAT-2 and
are required for preventing Ad-mediated induction of CRACC receptor on APCs. For this, a
variety of pharmacological inhibitors were used and the expression of CRACC receptor
following Ad-EAT2 or Ad-Null infection was evaluated. RAW264.7 cells were pre-treated with
pharmacological inhibitors that block the Src-kinase pathway (PP2), the PI3K pathway
(wortmannin), the PLCγ pathway (U-73122), and the ERK MAP kinase pathway (PD98059)
prior to Ad infection, and the expression of CRACC receptor was evaluated by quantitative RTPCR at 15 hpi. Inhibition of the Src-kinase or the PI3K signaling pathways did not affect the
ability of EAT-2 to prevent Ad induced CRACC receptor expression (Figure 46A). However,
blocking the ERK MAP Kinase signaling pathway by use of the MEK kinase inhibitor PD98059,
or the PLCγ signaling pathway by the use of U-73122, completely eliminated the ability of AdEAT-2 to prevent Ad-mediated induction of CRACC receptor expression in RAW264.7 cells
(Figure 46B).
Figure 46: EAT-2 requires functional ERK and PLCγ pathways to down-regulate CRACC
receptor on APCs. CRACC receptor expression in RAW264.7 cells at 15 hpi following AdEAT2 (black) or Ad-Null (gray) (MOI of 20,000) in the presence or absence of the following
inhibitors: PD98059 (5 µM), U-73122 (1 µM), PP2 (1 µM), and Wortmannin (5 µM). (A)
CRACC receptor expression in the presence or absence of Src-kinase or PI3K signaling
pathways inhibitors. (B) CRACC receptor expression in the presence or absence of ERK or
PLCγ signaling pathways inhibitors. Statistical analysis was completed using Two-Way
192
ANOVA with a Bonferroni post-hoc test p<0.05 was deemed a statistically significant
difference. * denotes p<0.05, ** denotes p<0.001 statistically different from mock infected cells.
# denotes p<0.01, ## denotes p<0.001, significantly different from Ad-infected kinase inhibitors
non-treated cells.
193
Figure 46: EAT-2 requires functional ERK and PLCγ pathways to down-regulate CRACC
receptor on APCs.
194
To further confirm the role of a functional ERK MAPK signaling pathway in EAT-2
mediated prevention of CRACC receptor up-regulation in Ad infected RAW264.7 cells, we
examined ERK phosphorylation in Ad infected cells. Consistent with our previously published
work (421), Ad infection of RAW264.7 cells induced ERK phosphorylation as early as 4 hpi. At
4, 8, and 12 hpi, no significant differences were observed between Ad-EAT2 and Ad-Null
infected cells (Figure 47A). Importantly, at16 and 24 hpi, Ad-EAT-2 induced higher ERK
phosphorylation compared to Ad-Null infected control (Figure 47A and B).
Figure 47: EAT-2 over-expression induces ERK phosphorylation in RAW264.7 cells.
Western blot analysis for ERK phosphorylation following Ad-EAT2 or Ad-Null infection at the
indicated time points was performed. Phosphorylated Erk1/2 and Erk2 levels were determined
as described in materials and methods using LI-COR Odyssey. To control for loading,
quantification was performed after normalizing the pErk1/2 to Erk2 levels. A two-tailed
homoscedastic Student’s t-test was used to calculate differences. p<0.05 was deemed a
statistically significant difference.
195
Figure 47: EAT-2 over-expression induces ERK phosphorylation in RAW264.7 cells.
196
DCs and macrophages express most of the SLAM family receptors. (422) Since the
SLAM (CD150) receptor is involved in the regulation of cytokine secretion by human and mouse
macrophages and dendritic cells (292) and the expression of these mediators enhanced the
adaptive immune responses to vaccine antigens, we sought to evaluate a possible role for EAT-2
over-expression to augment the secretion of cytokine/ chemokine directly from macrophages.
ERK MAPK dependence of these responses was also investigated. RAW264.7 cells were
infected with Ad-EAT2 or Ad-Null and the concentration of multiple cytokines and chemokines
present in the overlying medium was determined. Utilizing this system and multiplicity of
infection, minor inductions of some cytokines and chemokines were quantified following Ad
infection (Figure 48). Interestingly, Ad-EAT-2 infection of these cells induced a more robust
secretion of cytokines and chemokines as compared to Ad-null infected cells (Figure 48).
Specifically, EAT-2 over-expression significantly (p<0.001) induced higher levels of G-CSF,
MIP-1β, IL-13, Eotaxin, and TNFα, as compared to Ad-Null infected cells (Figure 48).
RAW264.7 cells pre-treated with PD98059 prevented EAT2 over-expression from inducing the
secretion of TNF-α, G-CSF, and IL-6 (p<0.001) (Figure 48A). Conversely, PD98059 pre-treated
RAW264.7 cells had an enhanced secretion of MIP-1β and IFNγ when infected with Ad-EAT2
as compared to infection with the Ad-null control virus (Figure 48B). In addition, RAW264.7
cells pre-treated with PD98059 partially prevented EAT2 over-expression from inducing the
secretion of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17,
Eotaxin, KC, and GM-CSF (Figure 48C).
Figure 48: EAT-2 is a critical cytokine and chemokines regulator in macrophages.
RAW264.7 cells were mock infected or infected with Ad-EAT2 or Ad-Null for 72 hours at MOI
of 20,000 in the presence or absence of ERK inhibitor (PD98059). Culture media was collected
197
at 72 hours post-Ad infection and was analyzed for cytokines and chemokines levels using a
multiplexed bead array based quantitative system. Statistical analysis was completed using a
one-way ANOVA with a Student-Newman-Keuls post-hoc test. Samples were plated in
quadruplicate and are expressed as mean ± SD. *, *** -Indicate cytokine values that are
statistically different from those in mock infected cells, p<0.05, p<0.001 respectively. #, ##, ###,
indicate cytokine values that are statistically different from those in Ad-infected PD98059 nontreated cells, p<0.05, p<0.01, p<0.001 respectively.
198
Figure 48: EAT-2 is a critical cytokine and chemokines regulator in macrophages.
199
5.3.
Discussion
The development of advanced generation vaccines will be required to eradicate those
pathogens still plaguing mankind. To develop these vaccines to their fullest potential will require
in depth understanding of the ability of the innate immune system to facilitate induction of
robust, antigen specific adaptive immune responses. Numerous systems are being evaluated in
this regard, and each has benefits or limitations that will need to be fully ascertained before
progress will be made in the elimination of diseases such as HIV-AIDS, malaria, and
tuberculosis. We have investigated several vaccine platforms, and discovered that Ad mediated
transduction of the SLAM family of receptors adaptor EAT-2 allowed for improved induction of
antigen specific adaptive immune responses to Ad expressed antigens (103).
In an effort to further understand the molecular mechanisms underlying this important
discovery, we have shown here that use of Ad vectors in general results in the upregulation of the
SLAM family member CRACC, a response that may limit the full potential of Ad vectors to
induce maximal, antigen specific adaptive immune responses. This insight is provided by our
results demonstrating that EAT-2 over-expression during Ad mediated antigenic gene transfer
specifically prevents Ad induction of CRACC. This ability is at least dependent upon a
functional ERK MAPK pathway, as well the potential interaction between the EAT-2 SH-2
domain and the phosphorylated ITSMs of SLAM receptors. The phenomenon of reduced
CRACC receptor expression on APCs has been associated recently with increased antigeninduced CD4+T cells proliferation, as well, IL-2 and IFNγ, production, consistent with our
recently published work (151, 191).
200
The SLAM family of receptors is an emerging family of immune receptors that regulate
the functions of both innate and adaptive immune cells (410). CRACC is a novel member of the
SLAM family (423, 424) and is expressed on human plasma cells, NK cells, activated CD8+ T
cells, activated B cells, macrophages, and dendritic cells (422, 424). Our results demonstrate that
in vivo, Ad-EAT-2 transduced APCs function as a more potent APC as compared to
conventional Ad transduced APCs, and this activity is correlated with a reduced abundance of
cell surface CRACC receptor. This phenomenon may diminish CRACC-CRACC interactions at
the immunological synapse between Ad-EAT2 transduced DCs and potentially responsive naïve
T cells, and based upon these and our previous results, results in improved induction of antigen
specific adaptive immune responses (103). Decreased CRACC-CRACC interactions between
EAT-2 over expressing APCs and naïve T cells may limit CRACC activation and
phosphorylation within the T cells, preventing binding of inhibitory phosphatases to the
phosphorylated ITSMs of the T cell CRACC receptor, and thereby enhancing the induction of
robust antigen specific, T cell based adaptive immune responses. Whether or not other vaccine
platforms also increase CRACC expression is a question that our studies suggests should be
addressed, as inhibition of this response may also facilitate improved effectiveness of those
platforms as well.
Alternatively (though not mutually exclusive), increased CRACC receptor expression on
stressed cells has been associated with enhanced targeting of the cells for destruction by NK cells
(150, 151). Cells transduced by first-generation Ad vectors are known to be targeted for
elimination by NK cells (193, 425). Thus, decreased abundance of cell surface expression of
CRACC by Ad-EAT2 transduced dendritic cells and macrophages may make them less
201
susceptible to CRACC-mediated NK cell mediated cytotoxicity, allowing for an improved
potential to induce antigen specific adaptive immune responses.
Previous reports have shown that the SLAM (CD150) receptor is involved in the
regulation of cytokine secretion by human and mouse macrophages and DCs (292). Our findings
here have complemented that work by demonstrating involvement of the SLAM family receptors
adaptor EAT-2 in the secretion of multiple cytokine and chemokines, thereby linking SLAM
signaling by EAT-2 to the mediation of cytokines and chemokines production in macrophages.
Furthermore, a recent report demonstrated that activation of the SLAM family member CD84
enhanced the secretion of TNFα and MCP-1 by mouse RAW264.7 and bone marrow derived
macrophages following TLR4 activation (426). This may suggest that the enhanced cytokine and
chemokines profile after Ad-EAT-2 infection could be as a result of synergy between SLAM
receptors signaling by EAT-2 and activation of TLR4, or other TLRs, signaling by Ads (8, 184).
The enhanced production of these cytokines could also act as autocrine growth and
differentiation factors as well as positive modulators of the immune responses by both DCs and
macrophages. Further, autocrine stimulation by TNFα regulates the expression of many proinflammatory response genes, as well prime macrophages for enhanced responsiveness to
cytokines and inflammatory stimuli (427). Production of TNFα after Ad-EAT2 infection was
several orders of magnitude higher than its production after Ad infection. Thus, the present
evidence supports the additional involvement of TNFα in Ad-EAT2 mediated–increases in
cytokine expression. It is possible that other highly induced cytokines, such as G-CSF, IL-1β,
and MIP-1β, also play a role in EAT-2 function in macrophages as well.
Previous reports have shown that activation of CRACC recruits the EAT-2 adaptor,
resulting in activation of the phospholipase Cγ signaling pathway in NK cells (150). Our results
202
suggested that the negative regulation of the CRACC receptor and the induction of several
cytokines and chemokines by APCs after Ad mediated overexpression of EAT-2 are also
mediated by a mechanism(s) that requires functional PLC-γ and ERK MAP kinase signaling
pathways. Finally, since EAT-2, but not SAP binds to the CRACC receptor (150, 151), the
reduced cell surface expression of CRACC in Ad-EAT2 transduced APCs may result in
enhanced triggering of a negative feedback mechanism regulating CRACC receptor overall.
These results, combined with our previous work (103, 191, 356), allows us to
hypothesize a model for EAT-2 molecular mechanism in APCs (Figure 49). In this model, we
hypothesize that upon EAT-2 binding to the phosphorylated ITSMs of SLAM family receptors
results in recruitment of effector molecules in the phosphorylated C-tail of EAT-2 and the downstream activation of PLC-γ and the ERK signaling pathways, enhanced transcription of various
cytokines and chemokines genes, as well as down-regulation of the CRACC receptor from the
APC surface. Furthermore, augmenting SLAM signaling by EAT-2 induces higher levels of
CD80 and CD86 co-stimulatory molecules, CD40, and MHC-II molecules on the APC surface
that enhances transgene specific T cells proliferation and IFN-γ and IL-2 production (191, 356,
428).
Figure 49: Model of EAT-2 molecular mechanism in APCs. In presence of high abundance of
EAT-2 adaptor, EAT-2 binds to the phosphorylated ITSMs of SLAM family receptors and as a
result the tyrosine residues at the EAT-2 C-terminal tail get phosphorylated. The phosphorylated
tyrosine on EAT-2 C-tail serves as a docking site for down-stream, yet un-identified, mediators
that transmit positive signals that result in down-stream activation of PLC-γ and ERK signaling
pathways, enhanced transcription of various cytokines and chemokines genes, as well as downregulation of the CRACC receptor from the APC surface. Furthermore, augmenting SLAM
203
signaling by EAT-2 induces higher levels of CD80 and CD86 co-stimulatory molecules, CD40,
and MHC-II molecules on the APC surface that enhances transgene specific T cells proliferation
and IFN-γ and IL-2 production. In T cells, activation of CRACC receptor enhances the
recruitment of inhibitory phosphatases that attenuate TCR-mediated activation.
204
Figure 49: Model of EAT-2 molecular mechanism in APCs.
205
In summary, we have determined that Ad vector mediated transduction of antigen
expressing transgenes results in upregulation of CRACC on the surface of APCs. This effect
inhibits the induction of maximal antigen specific adaptive immune responses by the Ad vector
platform. Simultaneous overexpression of EAT-2 by Ad vectors prevents CRACC receptor
upregulation on dendritic cells and macrophages improving the induction of proinflammatory
cytokine and chemokine responses, maturation of APCs, and the induction of improved, antigen
specific adaptive immune responses by Ad based vaccines. Future studies are needed to
determine if other vaccine platforms can be similarly improved by modifying SLAM receptorSAP adaptor interactions.
Acknowledgements:
We wish to thank the Michigan State University Laboratory Animal support facility for
their assistance in the humane care and maintenance of the animals utilized in this work. A.A.
was supported by the National Institutes of Health grants RO1DK-069884, P01 CA078673, the
MSU Foundation as well the Osteopathic Heritage Foundation. YAA was supported by the King
Abdullah bin Abdulaziz Scholarship, Ministry of Higher Education, Kingdom of Saudi Arabia.
Author Disclosure Statement: No competing financial interests exist.
206
Chapter VI
Materials and Methods
207
6.1.
Adenovirus vector construction
All novel Ad vectors were constructed utilizing pAdEasy based system (429) with
modifications. A first-generation, human Adenovirus type 5 derived replication deficient vector
(deleted for the E1 and E3 genes) encoding EAT-2, HIV/Gag, CSP, rEA, and GFP as a transgene
were used in these studies. Infectious titers of all Ads were determined by standard Tissue
Culture Infectious Dose 50 (TCID50) method (AdEasy Adenoviral vector system manual,
Qbiogene, Carlsbad, CA). Infections titer was calculated by using KARBER statistical method:
1 + d(S-0.5)
TCID50/ml titer = 10 × 10
, where d is the log (10) of the dilution and S is the sum of
ratios from the first dilution. All viruses were found to be RCA free both by RCA PCR (E1
region amplification) and direct sequencing methods. Ad vectors have also been tested for the
presence of bacterial endotoxin as previously described (430) and were found to contain <0.01
EU per injection dose.
6.1.1. EAT-2 expressing Ads construction:
The Open Reading Frame of EAT-2 gene (Genbank Accession #NM_012009),
http://www.ncbi.nlm.nih.gov/nuccore/148747581, was excised using primers flanked by XhoI
and XbaI restriction endonucleases (NEB, Ipswich, MA) from a plasmid (Open Biosystem,
Huntsville, USA) and subcloned into the pShuttle vector which contains a CMV expression
cassette. The resulting pAdTrack-EAT-2 shuttle plasmid was linearized with PmeI restriction
enzyme and homologously recombined with the pAdEasyI Ad5 vector genome as previously
described (429) yielding pAd-EAT2. HEK293 cells were transfected with PacI linearized
plasmid and viable virus was obtained and amplified after several rounds of expanding infection.
Ad-EAT2 virus was purified using a CsCl2 gradient as previously described (431). The titer
208
obtained was approximately 2.3 × 10
12
vp/ml. direct sequencing and restriction enzyme mapping
were carried out to confirm the integrity of the EAT-2 sequence.
6.1.2. Ad-HIV/Gag construction:
To construct Ad-HIV/Gag, the HXB2 Gag gene (Genbank Accession #K03455),
http://www.ncbi.nlm.nih.gov/nuccore/1906382, was blunt end sub-cloned into the EcoRV site of
pShuttle-CMV. Restriction digests and sequencing were used to confirm the sequence integrity
and correct orientation of the resulting shuttle (pShuttle-CMV Gag). Recombination and viral
propagation was completed as described above.
6.1.3. Ad-CSP construction:
The Open Reading Frame (ORF) of the P. falciparum CS protein gene, composed of a
codon optimized consensus of several P. falciparum CS protein sequences (Figure 16), was
incorporated into plasmid pGA4 (GENEART, Burlingame, CA) and excised from pGA4 using
endonuclease NheI (NEB, Ipswich, MA). The excised portion was subcloned into the pAd
Shuttle vector containing a CMV expression cassette. Recombination and viral propagation was
completed as described above. Direct sequencing and restriction enzyme mapping were carried
out to confirm the fidelity of the CS protein sequence.
6.1.4. Ad-GFP and Ad-GFP/rEA construction:
The Open Reading Frame of rEA gene was inserted into an identical CMV driven
cassette directly upstream of the GFP cassette. The resulting pAdTrack-rEA shuttle with the
pAdEasyI Ad5 vector genome was prepared and constructed as described above yielding AdGFP/rEA. Direct sequencing and restriction enzyme mapping were carried out to confirm the
integrity of the rEA sequence. A direct comparison of transduction efficiency was completed for
209
Ad-GFP and Ad-GFP/rEA virus preparations using flow cytometry. Similar transduction
efficiencies were observed.
6.2.
Validation of viral particles (VP) titers of Ads
6.2.1. Silver Staining
To verify that particle number quantification was accurate across all Ads constructed,
10
10
vps of lysed purified virions of each Ad were separated by 10% SDS-PAGE and
subsequently stained with silver nitrate utilizing a Silver stain kit for proteins (Sigma, St. Louis,
MO). The amount of hexon protein was quantified for each Ad vector by scanning densitometry
using ImageJ software, ver. 1.29 (developed at the U.S. National Institutes of Health and
available on the Internet at http://rsb.info.nih.gov/nih-image/). Results from this analysis
indicated that the VP titers of all viruses determined by spectrophotometry fall within ~1.1 fold
of each other.
6.2.2. Western Blotting
To further verify that particle number quantification was accurate across all Ads
constructed, 1010 of lysed purified virions of each Ad were separated by 10% SDS-PAGE and
Western blotting was performed utilizing hexon specific antibodies (Abcam, Cambridge, MA).
Electrophoretically separated capsid protein samples were transferred onto nitrocellulose
membranes and probed with rabbit polyclonal Ad5 hexon specific antibody, followed by probing
with a fluorescent secondary antibody as previously described (184). Membranes were scanned
and hexon concentrations quantified using Licor’s Odyssey scanner (184). Results from this
analysis indicated that the VP titers determined by spectrophotometry fall within ~1.3 fold of
each other based on this assay, thus Ad5 vector preparations did not contain less virions as
compared to conventional first generation Ad5 vectors based on this assay (data not shown).
210
6.3.
Animal procedures
All animal procedures were reviewed and approved by the Michigan State University
ORCBS and IACUC. Care for mice was provided in accordance with PHS and AAALAC
standards (ID number: A3955-01). Adult male C57BL/6 and Balb/c mice were purchased from
The Jackson Laboratory (Bar Harbor, ME). Intravenous injection of animals (8-10 weeks old)
consisted of injection (via the retro-orbital sinus) of 200µl of a phosphate-buffered saline
solution (PBS, pH 7. 4), containing 7.5 × 1010 total virus particles after performing proper
anesthesia with isofluorane. Plasma and tissue samples were obtained and processed at the
indicated times post-injection as previously described (421). Intramuscular injections were
completed by injection of the indicated virus particles in a total volume of 20 µl into the tibialis
anterior of the right hind limb. All procedures with rAds were performed under BSL-2, and all
vector treated animals were maintained in ABSL-2 condition. Care for mice was provided in
accordance with PHS and AAALAC standards.
Three groups of mice were analyzed in Ad-EAT2 and Ad-HIV/Gag co-injection study:
C57BL/6 or BALB/cJ mice were mock-injected with PBS, control group injected with Ad5HIV/Gag +Ad-GFP, and the experimental group injected with Ad5-HIV/Gag+ Ad-EAT2.
Control and experimental mice were sacrificed at different times after mock or virus treatment.
Three groups of mice were analyzed in Ad-EAT2 or Ad-GFP/rEA and Ad-CSP coinjection study: BALB/cJ mice were mock-injected with PBS, control group injected with Ad5CSP +Ad-GFP, and the experimental groups injected with Ad5-CSP+ Ad-GFP/rEA or Ad5-CSP
+Ad-EAT2. Control and experimental mice were sacrificed at different times after mock or virus
treatment.
211
Four groups of mice were analyzed for rEA molecular mechanism study. MyD88-KO
and TRIF-KO mice were kindly provided by Dr. Shizuo Akira. MyD88/TRIF-DKO mice were
bred at Michigan State University. Intraperitoneal (IP) injection of animals (2-4 months in age)
consisted of 100µl phosphate-buffered saline solution (PBS, pH 7.4) containing 100 ng rEA
protein from Eimeria tenella as previously described (133, 386). rEA protein purification was
performed as previously described (131) with minor modifications as described (131, 133).
Plasma and tissue samples were obtained and processed at the indicated times post-injection as
previously described (133). Importantly, IP route of rEA administration was confirmed to be
more efficient that other widely used routes for adjuvant injection, such as intranasal or
subcutaneous. To study rEA-triggered activation of immune cells by flow cytometry and bead
array methods, plasma and spleen tissues were harvested at 6 hours post injection (hpi), whereas
for studies measuring activation of signaling pathways the spleen and liver tissues were
harvested at 0, 20, 40, 60, 90, and 120 minutes post-rEA injection.
6.4.
Cytokine and chemokine analysis
Proinflammatory cytokines/chemokines in the murine plasma collected in vivo or in the
media collected from cultured DCs or RAW264.7 cells were measured utilizing a 7- or 23-plex
multiplex based array system. Plasma samples were collected using heparinized capillary tubes
and EDTA coated microvettes (Sarstedt, Nümbrecht, Germany) and centrifuged at 3400 rpm for
10 min to retrieve plasma samples. All procedures were performed exactly as previously
described per the manufacturer’s instructions (Bio-Rad, Hercules, CA) via Luminex 100
technology (Luminex, Austin, TX) as previously described (133).
6.5.
Quantitative RT-PCR Analysis
212
To determine relative levels of a specific, liver or spleen derived RNA transcript, corresponding
tissues were snap frozen in liquid nitrogen and RNA was harvested from ≈100mg of frozen
tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) per the manufacturer’s protocol.
Following RNA isolation, reverse transcription was performed on 180ng of total RNA using
SuperScript II (Invitrogen, Carlsbad, CA) reverse transcriptase and random hexamers (Applied
Biosystems, Foster City, CA) per manufacturer’s protocol. RT reactions were diluted to a total
volume of 60µl and 2µl was used as the template in the subsequent PCR reactions. Primers were
designed using Primer Bank web based software (http://pga.mgh.harvard.edu/primerbank/).
Complete list of primers utilized in this study is available in table 3. Quantitative PCR (qPCR)
was carried out on an ABI 7900HT Fast Real-Time PCR System using SYBR Green PCR
Mastermix (Applied Biosystems) in a 15 µl reaction. PCRs were subjected to the following
procedure: 95.0 1C° for 10 min followed by 40 cycles of 95.0 1C° for 15 s followed by 60.0
1C° for 1 min. The comparative Ct method was used to determine relative gene expression using
GAPDH to standardize expression levels across all samples. Relative expression changes were
calculated based on comparing experimental levels of a respective spleen transcript to those
quantified in spleen samples derived from mock injected animals.
213
Table 3: List of primers, utilized in qRT-PCR experiments. A pair of Forward (For) and
Reverse (Rev) primers is provided for every transcript tested by qRT-PCR based methods. The
primers were designed as described above; the length of resulted PCR products was 100-160
nucleotides.
Gene
Forward primer (5' -> 3')
Reverse primer (5' -> 3')
EAT-2
CTGGGACTGATCTCAGGGTG
GAAGGGAACGGGAGAATGGG
CRACC
CCGACTTGTGCCCTCACTTAG
GAGCTGGGACTCTTTACCACT
2B4
CTCGGGGCCATCATTTGTTTC
GCTAGAAGGGAGCTGAACATCA
SLAM
AAATCAGGGGTACGTTCTATGC
TCCTGTGCGAAATATGACAGAC
Ly108
TCTCCAGGGAACACTGTGTATG
GGTTGGTTATAGCCGGTTAAAGC
Ly9
TCAGGGATGCTAGGGGGTTC
TTCGCTGACTTTGAGTCTGCC
CD84
TTATTCTCATTCCGATGTTGGCA
GTGGGTTGAGCATTTCTTGAAAC
NOD-1
CCCCTTCCCAGCTCATTCG
TGTGTCCATATAGGTCTCCTCCA
TNFa
CCCTCACACTCAGATCATCTTCT
GCTACGACGTGGGCTACAG
ADAR
AGGATTGGTGAGCTCGTCAG
GCCCTGTTTCTTGCTGTGTG
ICAM-1
GGCATTGTTCTCTAATGTCTCCG
GCTCCAGGTATATCCGAGCTTC
IL-6
TAGTCCTTCCTACCCCAATTTCC
TTGGTCCTTAGCCACTCCTTC
IL-15
TCTCCCTAAAACAGAGGCCAA
TGCAACTGGGATGAAAGTCAC
6.6
Isolation of Splenocytes
Splenocytes from individual mice were harvested and processed by physically disrupting
and facilitating passage of splenic tissue through a 40 µm sieve, followed by induction of RBC
lysis by using 2 ml of ACK lysis buffer (Invitrogen, Carlsbad, CA) per homogenized spleen.
214
Splenocytes were subsequently washed two times with RPMI medium 1640 (Invitrogen,
Carlsbad, CA) supplemented with 10% FBS, 2 mM L-glutamine, and 1% PSF (penicillin,
streptomycin, fungizone), then resuspended and counted using the automated cell counter
“countess” (Invitrogen).
6.7.
Cell staining and flow cytometry
Splenocyte preparations were evaluated for the presence of DC, macrophages, NK, NKT,
6
T, and B cell activation/maturation markers as previously described (172). 2×10 cells were
stained with combinations of the following antibodies: APC-CD3, PerCpCy5.5-CD19, PE-Cy7NK1.1, and PE-CD69 (all 4 µg/ml) or PECy7-Cd11c, APCCy7-CD11b, APC-CD80, Pacific
Blue-CD86, Alexa Fluor700-MHCII, FITC-CD40. For DCs and macrophages analysis, a dump
channel had been applied using PerCpCy5.5-CD3/CD19/NK1.1 (all 4 µg/ml) antibodies (BD
Biosciences, San Diego, CA). Cells were incubated on ice with the appropriate antibodies in
2.4G2 hybridoma cell supernatant for 30 minutes, then washed and sorted using a BD LSR II
6
instrument. For NK/NKT cells IFNγ intracellular staining, 3×10 splenocytes were incubated
with Brefeldin A (1 µg/ml) in DMEM with 10%FBS/1xPSF for 4 hours at 37 ° C. Following
incubation, splenocytes were washed two times with FACS buffer, incubated for 15 minutes with
purified rat anti-mouse CD16/CD32 Fcγ block (BD Biosciences, San Diego, CA), surface
stained with CD3-APC and NK1.1-PECy7 (8 µg/ml) for 30 minutes at 4 C, washed with FACS
buffer, fixed with 2% formaldehyde (Polysciences, Warrington, PA) for 20 minutes on ice,
permeabilized with 0.5% Saponin (Sigma-Aldrich, St. Louis, MO) for 20 minutes at room
temperature, and incubated on ice with IFNγ-FITC (8 µg/ml) for 2 hours. Samples were analyzed
on a BD LSR II instrument using FlowJo software (Tree Star, San Carlos, CA, USA). For
215
Adenovirus cell transduction, splenocytes were subjected to flow cytometry and the percentages
of Ad-GFP transduced cells (FITC+) were calculated using FlowJo software.
For BMDMs and RAW264.7 cells studies, the following antibodies were used: APCCD80, V450-CD86 (BD Biosciences, San Diego, CA), Alexa Floure700-MHC-II, and FITCCD40 (eBioscience, San Diego, CA) and CRACC-APC (cat# FAB4628A) (R& D System).
For 7-color intracellular cytokines staining, cells were surface stained, fixed with 2%
formaldehyde (Polysciences, Warrington, PA), permeabilized with 0.2 % Saponin (SigmaAldrich, St. Louis, MO), and stained for intracellular cytokines. Violet fluorescent reactive dye
(ViViD, Invitorgen) was included as a viability marker to exclude dead cells from the analysis.
For tetramer staining, blood was isolated by retro-orbital bleeds and PBMCs were
isolated using Lympholyte-Mammal (Cedarlane, Burlington NC). Tetramer staining of PBMCs
was completed using a PE conjugated MHC-I tetramer folded with the AMQMLKETI (HIV/Gag
immunodominant epitope) or NYDNAGTNL (amino acids 43-51 of the CS protein sequence)
peptide generated at the NIH Tetramer Core Facility.
6.8.
In vitro cell culture:
Bone marrow cells were extracted from the femurs and tibiae of male 6-8 weeks old
Balb/c mice, and red cells were removed using ACK lysis buffer (Invitrogen, Carlsbad, CA).
Bone marrow cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) and 30% supernatant derived from confluent L929 cell
cultures. At day 7, immature macrophages were collected and plated into 12-well plates for 24
hours. This procedure yields a pure population of macrophage colony-stimulating factordependent, adherent macrophages. Murine RAW264.7 macrophages (ATCC TIB71) were
maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin–
216
5
streptomycin following standard procedures. Suspensions of 5 ×10 cells were seeded into each
well of 12-well plates. Then, the cells were incubated with 1 ml culture medium alone, or with
medium containing Ad-EAT2, Ad-Null or Ad-GFP at the indicated MOIs and time points.
For Pharmacological Inhibitors treatments, the following inhibitors were used: PP2 (1
µM), wortmannin (5 µM), PD-98059 (5 µM), and U-73122 (1µM) (all from BIOMOL).
6.9.
Murine CD11c+ DCs isolation
+
Mouse DCs (CD11c splenic cells) were isolated from Balb/c or C57BL/6 mice using the
magnetic assisted cell separation (MACS) system purchased from Miltenyi Biotech (Auburn,
CA) utilizing the established protocols from the manufacturer. DC sorting by flow cytometry
resulted in >95% pure CD11c positive cell population. The viability of recovered DCs was ~95%
+
as measured by trypan blue viability staining. Isolated CD11c cells were seeded at a density of
5
0.3×10 cells per well into 24-well plates in 300 µl/well complete medium (DMEM/F12
supplemented with 10% FCS and gentamicin (10µg/ml). Mouse DCs were infected with AdEAT2 or Ad-Null (MOIs of 5000) for 72 hpi at 37 C in 5% CO2 and 95% ambient air.
Following incubation, cells were harvested and the expression of CRACC receptors was
evaluated by flow cytometry.
6.10. Murine IL12p70 measurement by ELISA from isolated CD11c+ DCs
+
For mIL12p70 production from isolated CD11c DCs, an in vitro bioassay originally
developed to monitor rEA activity during its purification and production, as well as provide
+
some insight into the mechanism of action of rEA, was previously described (131). CD11c DCs
were isolated from C57BL/6 WT, MyD88-KO, TRIF-KO, or MyD88/TRIF-DKO mice. Cells
217
5
were seeded at a density of 0.3×10 cells per well into 96-well plates in 200 µl/well complete
medium. The complete medium consisted of DMEM/F12 supplemented with 10% FCS,
gentamicin (10 µg/ml), recombinant mouse GMCSF (1 ng/ml), recombinant mouse IL4 (1
ng/ml), recombinant mouse IFN (3 ng/ml), and an agonistic anti mouse CD40 antibody (0.5
mg/ml) (R&D Systems, Minneapolis, MN). This synergistic combination of cytokines has no
significant effect on mouse IL12 release, but dramatically enhances the inducing effects of rEA
-6
1
on mouse IL12 release (131). Mouse DCs were stimulated with rEA (10 -10 ng/ml) overnight
(~18 hours) at 37 C in 5% CO2 and 95% ambient air. To study the specificity of TRIF inhibitory
effects in DCs, various toll-like receptor agonists were added to mouse DCs, using the following
concentrations: rEA (Barros Research Institute, Holt, MI, 100 ng/ml); E. coli 0111.B4 LPS (20
g/ml); R848 (0.5 g/ml) and ODN2006 (2.5 M). LPS, R848 and ODN2006 (TLR4, TLR7/8
and TLR9 agonists respectively) were purchased from InvivoGen, San Diego, CA, reconstituted
with endotoxin-free water and diluted in culture media (DMEM/F12 + 10% FCS). Following
incubation, culture medium was analyzed for mouse IL12p70 levels using an ELISA kit and
following its enclosed instructions (R&D Systems, Minneapolis, MN) or for 23 mouse
cytokines/chemokines using multiplex system (Bio-Rad, Hercules, CA).
6.11. CD8+ T cells depletion Analysis
+
CD8 T cells were depleted from pooled splenocytes preparations using MACS beads
and LS columns per the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach,
+
Germany). % CD8- SFC = SFC CD8-dep/SFC CD8 ). Depletion was verified using FACS
analysis using APC-CD8a, and Pacific Blue-CD4 antibodies (BD Biosciences, San Diego, CA).
6.12. ELISPOT Analysis
218
ELISpots were performed in accordance to manufacturer’s protocol using the Ready-set Go IFNγ
or IL2 mouse ELISpot kit produced by eBiosciences (San Diego, CA). Splenocytes were
stimulated ex vivo with 4μg/mL of the >98% pure CS protein immunodominant peptide
NYDNAGTNL (amino acids 43-51 of the CS protein sequence) or HIV/Gag immunodominant
peptide AMQMLKETI peptide (GenScript Piscataway, NJ). A library of 15mers overlapping by
5 amino acids spanning the entire CS or proteins non-repeating region or HIV/Gag protein was
constructed and also used to stimulate splenocytes ex vivo (Biosynthesis Inc., Lewisville, TX).
Spots were counted and photographed by an automated ELISPOT reader system (Cellular
Technology, Cleveland, OH). Ready-set Go IFNγ and IL-2 mouse ELISPOT kits purchased from
eBioscience (San Diego, CA).
6.13. In vivo CTL Assay
For Ad-EAT2 and Ad-HIV/Gag co-injection study, Balb/c or C57Bl/6 mice were covaccinated with equivalent doses of Ad-HIV/Gag with either Ad-GFP or Ad-EAT2 (totaling 1×
7
9
10 vps for Balb/c and 1X 10 vps for C57Bl/6 mice). At 14 days, syngeneic splenocytes were
isolated and either pulsed with an irrelevant peptide specific to the Plasmodium falciparum
circumsporozoite antigen (NYDNAGTNL) or with the HIV-Gag immunodominant
AMQMLKETI peptide or QBI# 304796 for 1 hour at 37ºC. Irrelevant peptide pulsed cells were
subsequently stained with 1μM CFSE (CFSE
with 10μM CFSE (CFSE
amount of both CFSE
High
Low
Low
) while Gag-peptides pulsed cells were stained
). Naïve and immunized mice were injected with equivalent
and CFSE
High
stained cells via the retro-orbital sinus. After 5 hours,
mice were terminally sacrificed and splenocytes were recovered and sorted on an LSRII flow
cytometer. FlowJo software was used to determine percentages of CFSE stained cells. %
219
Specific killing = 1-((% CFSE
High
/ % CFSE
Low
) immunized / (% CFSE
High
/ % CFSE
Low
)
non-immunized). For Ad-EAT2 and Ad-CSP co-injection study, same procedures were performed
on Balb/c mice except that the in vivo CTL killing was evaluated 18 hours following the adoptive
transfer of NYD- or non-specific peptide (AMQ)-loaded splenocytes injection.
6.14. Detection of CSP antibody in murine serum by ELISA
ELISA-based antibody assays were completed as previously described (184). Highbinding flat bottom 96-well plates were coated with 0.2μg of purified CS protein per well in a
volume of 100μL and incubated overnight at 4C. Plates were washed with PBS-Tween (0.05%)
then blocked with blocking buffer (3% bovine serum albumin) for 1 hour at room temperature.
Plasma was diluted (1:50, 1:100, 1:200, 1:400) in blocking buffer and added to the wells and
incubated for 1 hour at room temperature. Wells were washed with PBS-Tween (0.05%) and
HRP antibody (Bio-Rad) was added at 1:4000 dilution in PBS-Tween. Tetramethylbenzidine
(TMB) (Sigma-Aldich) was added to each well and the reaction was stopped with 1N phosphoric
acid. Plates are read at 450nm in a microplate spectrophotometer. Subisotyping tittering was
completed with a hybridoma subisotyping kit (Calbiochem, La Jolla, CA) with plasma dilutions
of 1:50, 1:100. 1:200. 1:400. Statistical analyses were performed using Student t-test.
6.15. Western blotting
Spleen and liver tissues were homogenized in lysis buffer (20mM Tris-HCl, pH 7.4,
1mM EDTA, 150mM NaCl) containing 1% Triton X-100 with protease inhibitors. Homogenized
tissues were then centrifuged at maximum speed (13,000×G) for 10 min at 4°C, after which the
protein concentration of the supernatant determined using BCA method. Western blotting for
pErk1/2 and Erk2 was performed as previously described (184). Equivalent concentrations of
protein samples were run on polyacrylamide gels and transferred onto nitrocellulose membranes.
220
Blots were then probed with fluorescent antibodies as previously described, which included
antibodies against pErk1/2 (Cell Signaling, Inc., Boston, MA) and Erk2 (Santa Cruz
Biotechnologies, Santa Cruz, CA). Blots were scanned and bands were quantified using Licor’s
Odyssey scanner. For data analysis, the fluorescence of pErk1/2 bands was normalized to Erk2
bands prior to quantification.
6.16. Statistical analysis
For every experiment, pilot trials were performed with 3 mice per group (or N=3 for in
vitro experiments). This allowed us to determine effect size and sample variance so that Power
Analysis could be performed to correctly determine the number of subjects per group required to
achieve a statistical Power > 0.8 at the 95% confidence level. Statistically significant differences
in toxicities associated with innate immune response (i.e. proinflammatory cytokines, gene
induction, etc.) were determined using One Way ANOVA with a Student-Newman-Keuls posthoc test (p value < 0.05). Furthermore, a Two Way ANOVA with a Bonferroni post-hoc test was
used to analyze the levels of cytokines at 1 and 6 hpi (or other specified time points) to determine
significant differences (p value < 0.05) between groups. Statistically significant differences in
ELISpot assays were determined using One Way ANOVA with a Student-Newman-Keuls posthoc test (p value < 0.05). For ELISA analysis, a t-test was used to assess significance between
treatments. For multi-parameter flow cytometry, a One Way ANOVA with a Student-NewmanKeuls post-hoc test was used. For in vivo CTL assay, a One Way ANOVA with a StudentNewman-Keuls post-hoc test was used. Furthermore, a Two Way ANOVA with a Bonferroni
post-hoc test was used to analyze the effect of pharmacological inhibitors (p value < 0.05). For
western blot analysis, a two tailed Student’s t-test was used to compare two groups of virus-
221
infected cells. All graphs are presented as Mean of the average ± SD. GraphPad Prism software
was utilized for statistical analysis.
222
Chapter VII
Overall Summary and Significance
Agents that stimulate the innate immune system, generally referred to as immune
adjuvants, or simply adjuvants, have received attention as tools for designing more potent
vaccine platforms. Harnessing innate immunity therefore offers considerable promise in
designing the next generation of vaccine candidates. The molecular mechanism of action of
many adjuvants remained poorly understood. Several studies however have shown that
aluminum salts, the only FDA approved adjuvant, functions primarily by activating the innate
immune system by promoting recruitment and increasing antigen uptake by APCs, inducing
cytokine and chemokine secretions, and enhancing the expression of adhesion molecules
involved in migration of leukocytes (129).
Our studies in Chapters II and III have shown that, the Eimeria tenella derived protein,
rEA; possess all the properties of an ideal immunologic adjuvant. The rEA protein was
previously isolated from bovine small intestinal extracts and was shown to be a potent stimulator
of innate immune responses in various mouse models in vitro and in vivo, and to also have
remarkable anti-viral and anti-cancer activity (131). Administrations of rEA have been shown to
be safe and very well-tolerated in human clinical trials (432). We previously described a new
Ad-based vaccine vector that expresses the immunomodulatory TLR agonist, rEA (133). We
have proven that synergistic manipulation of TLR dependent innate immune responses by rEA
improve the ability of Ad-based vaccines to induce beneficial immune responses to pathogen
derived antigens, HIV-Gag (133). The molecular mechanism by which rEA functions as an
adjuvant is not completely understood, however our data suggested that rEA enhanced release of
223
pro-inflammatory cyto/chemokines from antigen presenting cells as well enhancing their
maturational characteristics may facilitate more efficient antigen processing and MHC class I
and II antigen presentation so as to improve antigen specific T cell (CD8+) responses. In
attempts to investigate the molecular mechanism underlying rEA adjuvant activity, we confirmed
that rEA rapidly activates multiple immune cell types, including DCs, macrophages, NK-, NKT-,
B-, and T-cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory
cytokines and chemokines responses, responses that completely depend upon the presence of the
TLR adaptor protein MyD88. During the course of this work, we were the first to identify that
the TRIF adaptor protein acts as a potent negative regulator of TLR (rEA) agonist-triggered
immune responses in mice. Our studies further confirmed that the TRIF suppressive activity was
not restricted to rEA-mediated responses, but were also apparent when TLR4 (LPS), TLR7/8
(R848) or TLR9 (ODN2006) agonists were used to stimulate DCs as well, thereby unveiling the
potential complexities of modulating TLR activity to augment vaccine efficacy. Future studies
could further expand upon these findings. For example, as our studies confirmed that DCs are the
main subset of innate immune cells that mediate rEA-triggered responses, future studies should
determine if ex vivo isolation of DCs and ex vivo exposure of the DCs to antigens and to rEA
could improve the potential use of rEA adjuvant in this DC-vaccine setting. Furthermore, rEA
has been shown to have adjuvant activity in human cells, which do not express the TLR receptor
that has been proposed for profilin molecules that are similar to rEA, such as TLR11/12. Future
studies will be required to investigate the innate immune receptor, as well as the innate immune
cell type, that rEA triggers to activate human immune cells.
The ability to easily scale up Ad5 vector production has resulted in thousands of patients
safely receiving recombinant, cGMP compliant, Ad5 based gene transfer vectors. The large
224
number of patients safely treated with the Ad5 platform in vaccine applications further supports
its high likelihood for acceptance by regulatory bodies, relative to less well tested platforms,
such as alternative Ad serotype derived vaccines, and/or other virus based vaccine platforms. Ad
vectors are being explored as potential vaccine candidates for a variety of pathogens. Reports
from experimental animal systems have shown that Ad5 vectors induce potent transgene
+
product-specific antibody and CD8 T cell responses in rodents, dogs, and nonhuman primates
(169, 433). For example, Ad vectors encoding a variety of antigens (such as, HIV/Gag, CSP, and
CEA) have an enhanced antigen presentation capacity and the ability to induce antigen specific
CTL responses (208). Further, E1 deleted Ad5 vectors expressing the HIV-1 gag, pol and nef
genes have been utilized in human trial (Merck® sponsored STEP trial). Despite the ability of
first generation rAd vectors to induce cellular and humoral immune responses in humans, the
results derived from the STEP trial suggest that a more potent vaccine capable of inducing
greater levels of antigen specific adaptive immune responses may demonstrate greater efficacy to
prevent HIV infection. These facts indicate a need for development of more efficacious Adbased vaccine vectors for this, and many other highly stringent clinical applications.
Several lines of investigation suggest that the induction of innate immune responses, (i.e.:
TLR-mediated signaling) may be an important reason as to why Ad based vaccines appear to be
superior to other vaccine platforms, especially in regard to elicitation of antigen specific cellular
immune responses. To describe the adjuvant characteristics of rAd5 vectors, we have reviewed
in detail the ability of rAd5 vectors to induce potent innate and adaptive immune responses to the
vector as well to the transgene it express. We have also reviewed several attempts to improve
rAd5-based vaccine platforms for the development of next generation vaccine candidates.
Utilization of advanced generation Ad vectors have been shown to allow for improved efficacy
225
in several vaccine based applications (434). In this dissertation, we have introduced a new
strategy for engineering rAd5 vectors, namely by enhancing beneficial innate immune responses
subsequent to rAd5 vaccination in an effort to improve the adaptive immune responses to the coadministered antigens. Specifically, we confirmed that targeted manipulation of intra-cellular
signaling initiated by the SLAM family of receptors, via Ad mediated co-expression of the
SLAM adaptor protein EAT-2 adaptor facilitated improved induction of several arms of the
innate immune system, and that these inductions positively correlated with an improved ability
of Ad vaccine formulations expressing EAT-2 to induce stronger cellular immune responses to
Ad vaccine expressed antigens. Ad5 vectors expressing EAT-2 facilitate bystander activation of
+
+
NK, NKT, B, CD4 , and CD8 T cells early after their administration into animals. EAT-2 overexpression also augments the expression of surface markers associated with the enhanced
function of antigen presenting cells, such as CD40, CD80, CCR7, and CD86. This multi-tiered
activation of the innate immune system by vaccine mediated EAT-2 expression also enhanced
the induction of Ad vaccine expressed antigens, such as the HIV-Gag antigen, as multiply
confirmed by Tetramer-based flow cytometry, ELISPOT, and in vivo CTL assays. Since both
mice and humans express highly conserved EAT-2 adaptors, our results suggest that human
vaccination strategies that specifically facilitate SLAM signaling may improve vaccine potency
when targeting HIV-1 antigens specifically, as well as numerous other vaccine targets in general.
In an innovative additional use of this novel vaccine platform, we utilized rAd5-EAT2
vaccine vectors for the development of rAd5-based vaccine that specifically target peptide
sequences derived from the malaria causing parasite, P. falciparum. Malaria is an infectious
disease that continues to devastate population world-wide, causing nearly 1 million deaths
annually, and morbidity that overwhelms the medical capabilities of developing countries. Some
226
of the most successful malaria vaccines studied to date have attempted to induce adaptive
immune responses to the P. falciparum CS protein, a protein that is found on the surface of P.
falciparum sporozoites and is also expressed by the parasite in hepatocytes during liver infection.
The induction of potent cellular immune responses to CS protein by a prophylactic malaria
vaccine could potentially eradicate both sporozoites and infected hepatocytes, preventing the
infection before clinical symptoms occur. In our studies to improve the immunogenicity of CS
expressing Ad vaccines, we confirmed the presence of a potent immunosuppressive activity for
the P. falciparum CS protein, when excessive TLR activation by advanced generation Ad
vaccines (Ad vaccine formulations expressing rEA) were utilized to enhance CS specific
adaptive immune responses. Future studies will need to be performed to elucidate the molecular
mechanisms as to how the CS protein suppresses immune responses to CS in the context of
excessive TLR activation. Our data suggest that the use of CS protein along with other
immunostimulatory compounds, such as TLR agonists, in certain malaria vaccine formulations
will have to be carefully considered.
In contrast, triggering SLAM family of receptors signaling by Ad vaccine mediated
expression of EAT-2 circumvented CS protein’s suppressive activity, and generated a potent
increase in the ability of Ad vaccines expressing CS to induce CS protein-specific T cell immune
responses.
Our findings suggest that augmentation of SLAM initiated signaling and downstream
inductions of specific cyto/chemokines may be the mechanism underlying EAT-2's ability to
improve induction of antigen specific CTL immune responses. The biochemical mechanism and
intracellular signaling pathway behind EAT-2's ability to function as a T cell stimulator is not
fully elucidated, but is a question that has been partially unveiled by our studies.
227
In Chapter V, we describe initial investigations as to the molecular mechanism
underlying how EAT-2 overexpression can facilitate improved induction of antigen specific
adaptive immune responses by Ad based vaccines. We found that EAT-2 over-expression
specifically prevents Ad vaccine induced CRACC receptor up-regulation on APCs, a
phenomenon that requires a functional ERK MAPK signaling pathway. We have also
demonstrated that, utilization of a mutated SH-2 domain form of EAT-2, EAT-2(R31Q), adaptor
failed to prevent Ad vaccine induced CRACC receptor up-regulation, suggesting a role for the
interaction between EAT-2 SH-2 domains and the phosphorylated ITSMs of SLAM family
members. The significance of EAT-2 to prevent Ad mediated up regulation of the CRACC
receptor on APCs needs to be further elucidated. Recently, it has been shown that the CRACC
receptor functions as an inhibitory molecule in T cells (144). This inhibitory activity is due to
homophilic CRAC-CRACC interactions between CRACC receptors on APCs and CRACC
receptors on T cells during their interactions, and specifically at the immunological synapse. This
homophilic interaction results in phosphorylation of the T cell CRACC receptor ITSMs, and
results in the recruitment of inhibitory phosphatases (SHP-1, SHP-2, and Csk) and SHIP-1 that
contain SH-2 domains. As a result of this inhibition, T cell proliferation, as well as IL-2 and
IFNγ secretion by the T cells in response to antigen presentations by APCs were abrogated.
Our studies suggest that expression of EAT-2 may have improved the adaptive immune
responses by preventing upregulation of CRACC by Ad vaccines transduced APCs, thereby
preventing T cell suppression by excessive CRACC stimulation. Future studies using isolated T
cells and DCs, coupled with specific inhibition of CRACC-CRACC interactions (i.e.: via use of
CRACC-blocking antibodies) could further validate these hypotheses. We also demonstrated that
EAT-2 over-expression regulates CRACC receptor expression at the transcriptional level,
228
suggesting the role for EAT2 responsive transcription factors. Future studies need to be
performed to elucidate this mechanism as well, such as CHIP array based analysis.
We also demonstrated that Ad mediated EAT-2 expression regulated the production of
several cytokines and chemokines, responses that were partially dependent upon the presence of
a functional ERK-MAPK signaling pathway. Future studies need to be performed to investigate
if one (or more) of these cyto/chemokines may be acting in an autocrine fashion to downregulate
CRACC expression in APCs, potentially via use of neutralizing antibodies or recombinant
proteins assays. Proving such a role would not only shed light on how EAT-2 signaling
facilitates CRACC down-regulation, but would also foster future studies to delineate the
mechanisms underlying regulation of CRACC expression in general.
We leave our readers with the view that the interactions between the innate and adaptive
immune system are multifaceted and complex. It is also clear that despite this level of
complexity, harnessing specific arms of the innate immune system, such as the SLAM or TLR
signaling dependent pathways, respectively, can have an important impact on the subsequent
induction of downstream, antigen specific adaptive immune responses. We predict a continued
expansion in the search for safe and efficacious vaccine adjuvants; as well inclusion of
pharmacological and/or other supportive interventions to further enhance both the safety and
efficacy of human vaccines.
229
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